The new commission opened at Westminster (4th Dec. 1568). The lords brought forward their charges against the queen accusing her of complicity in the murder of her husband. In proof of this they produced a number of letters that were supposed to have been contained in a casket left behind him by Bothwell in Edinburgh, when he fled from that city in June 1567. This casket contained eight letters and some sonnets, which, if really written by Mary, proved beyond doubt that she was hand in glove with Bothwell in bringing about the murder of Darnley. The Casket Letters considered in the light of her own conduct furnished damaging evidence of Mary's guilt. Whether these letters were genuine or forged is never likely to be established with certainty,[32] but considering the character of Mary's opponents, their well-known genius for duplicity, the contradictory statements put forward by their witnesses and the indecent haste with which the whole enquiry was brought to a close, it is difficult to believe that the evidence of Mary's authorship was convincing. The commissioners acting on Mary's behalf laboured under grave disadvantages from the fact that their mistress was not at hand for consultation. As a consequence they made many mistakes in their pleadings, but they were on sure ground when they demanded that copies of the incriminating letters should be forwarded to Mary for examination. This demand, though supported by the French ambassador, was refused, and Mary was never allowed an opportunity to reply to the main charge brought against her. An offer was made that proceedings should be dropped if Mary would consent to resign the throne of Scotland in favour of her son, and when she refused this offer the conference was brought to a sudden termination. Moray and his friends were informed that "nothing had been produced against them as yet that might impair their honour and allegiance; and on the other part there had been nothing sufficiently produced or shown by them against the queen their sovereign, whereby the Queen of England should conceive or take any evil opinion of the queen her good sister for anything yet seen" (Jan. 1569).[33] The Earl of Moray and his companions were allowed to return to Scotland, and nothing more was done either to establish the innocence or the guilt of the Queen of Scotland. The object of Elizabeth and her advisers had been attained. They had blackened the character of Mary; they had driven a wedge between herself and her nobles, and had allowed Moray to return to Scotland to rule as an English dependent.

To prevent Queen Mary from falling into the hands of the Catholic lords of the north she was removed from Tutbury to Coventry (26th January 1569). Whatever might be said of Mary's conduct during her early years in Scotland, or whatever doubt might have been entertained about her orthodoxy by the Pope and by the Catholic powers of the Continent, everything unfavourable to her was forgotten by them in their sympathy for her sufferings, and in their admiration for her fortitude and sincere attachment to her religion. Pius V. and Philip II. were as deeply interested in her fate as were the Catholics of Scotland and of England. A scheme was arranged to promote her marriage to the Duke of Norfolk and to secure her succession to the English throne, but Elizabeth anticipated the design by imprisoning the Duke, suppressing the rebellion of the northern lords (1569), and by braving the terrors of the papal excommunication levelled against her the following year.

When later on a new plot was discovered with the same object in view Norfolk was put to death (1572). While Mary was alive in England she was a source of constant danger to Elizabeth's throne. English Catholics driven to desperation by the penal laws were certain to turn to her as their lawful sovereign, while the Catholic nations on the Continent could fall back on the imprisoned queen whenever they chose to stir up disorder, or possibly to attempt an invasion. Dangerous as she was in prison, she might be still more dangerous if she were free to effect her escape either to Scotland or to France. In her death lay Elizabeth's best hope of peace, and as the rigour of her confinement failed to kill her, an attempt was made to induce the Scots to undertake a work that the English feared to undertake.[34] At last an opportunity was given of bringing about her execution and of covering the measure with an appearance of legality. A scheme for her release was undertaken by Babington,[35] with every detail of which the spies of Cecil were intimately acquainted, if they did not actually help to arrange them. Babington's letters to Mary and her replies were betrayed and copied. It is certain that Mary knew what was intended, but there is no evidence to show that she approved of the murder of Elizabeth. When the proper time came Babington and his accomplices were arrested and put to death (October 1586), and Mary's fate was submitted to the decision of Parliament. Both houses petitioned that the Queen of Scotland should be executed, but Elizabeth, fearful of the consequences and hoping that Mary's jailer Paulet, would relieve her of the responsibility, hesitated to sign the death warrant. At last, however, she overcame her scruples, and on the 8th February 1587, Mary Queen of Scots was beheaded at Fotheringay. Her attitude to the last was worthy of praise. She died a martyr for her religion, and by her death she expiated fully the imprudences and waverings of her youth. Elizabeth pretended to be horrified by the action of her ministers. Her secretary was imprisoned and fined to prove to Scotland, France, and Spain that the Queen of England had no responsibility for the tragedy of Fotheringay.

Meanwhile how fared it with Catholicism in Scotland? The Regent Moray returned from England early in 1569. Acting on the repeated requests of the General Assembly he undertook new measures against the Catholic Church. Catholic officials and professors were removed from Aberdeen University; several priests were arrested and punished though the regent was unwilling to inflict the death penalty, and many distinguished clerics and laymen, including the Primate and Bishop Leslie, were outlawed and their goods confiscated. The regent was not destined however to enjoy long the fruits of his treachery against his sister. In 1570, at the very time when he was plotting with the English government to get the Queen of Scotland into his power, he was shot in Linlithgow by one of the Hamiltons, the hereditary enemies of his house.

On his death there were two strong parties in Scotland. The majority of the nobles, including the Duke of Châtelherault, Argyll, Huntly, Atholl, and even Kirkcaldy and Maitland of Lethington, two former supporters of Moray, ranged themselves on the side of their imprisoned queen, and might have succeeded in re-establishing her authority had not Elizabeth espoused the cause of Morton, Mar, Glencairn and Ruthven, backed as these were by Knox and the preachers. Two English armies were dispatched into Scotland, and with the help of the English forces the Earl of Lennox, Darnley's father, was appointed regent (July 1570). It was not the first time that he had sought to destroy the independence of his country by invoking the assistance of the English, and as he had gone over to Protestantism he was determined to throw himself into the arms of the Reformers. The castle of Dunbarton was still in the possession of the queen's supporters. He laid siege to it, and captured it in April 1571. Here he seized the Primate of Scotland, and had him put to death after a summary trial. The chapter met and elected Robert Hay, but he was never consecrated, and for more than three hundred years St. Andrew's was without a Catholic bishop. In September 1571 Lennox was slain, and the Earl of Mar was elected regent. During his short reign he was unable to enforce his authority in the country. Negotiations were opened with him by Cecil's agents to induce him to undertake the execution of the Queen of Scotland, who was to be sent back from England for the purpose, but his sudden death in 1572 put an end to the scheme.

He was succeeded by the Earl of Morton, another of Elizabeth's agents. At first Morton was not unfavourable to the Catholics owing to the disputes that arose between himself and the preachers about the re-establishment of the episcopal form of government, but later on he adopted a policy of violent opposition to the old religion. Some of the priests were put to death; others were arrested or banished; a list of Catholics including Beaton the Archbishop of Glasgow, Leslie Bishop of Ross, and Chisholm Bishop of Dunblane was drawn up for proscription, and steps were taken to suppress Catholic holidays and to remove from the churches everything that called to mind Catholic devotions.

In 1578 the young king demanded Morton's resignation. A council of twelve was appointed in his place, at the head of which stood the Earls of Argyll and Atholl. Elizabeth was annoyed at the fall of her minion, and took no pains to conceal her annoyance from the young king. It looked as if friendly relations between the two courts might be broken, and the Catholic party both at home and on the Continent were filled with new hopes. In 1579 Esmé Stuart, Lord d'Aubigny, a nephew of the former Earl of Lennox, arrived from France, where he had been educated as a Catholic. He was welcomed at court by the king and created Earl of Lennox. James fell completely under his sway, though the preachers regarded d'Aubigny as a Catholic spy. Regardless of Elizabeth's friendship, James was induced to open communications with his mother, and when the Earl of Morton rose in rebellion against such a policy he was arrested and put to death (1582). Though apparently Lennox made profession of accepting the established religion in Scotland, he was endeavouring secretly to bring about an understanding between Mary and her son, to secure the release of the former from captivity, and to assist the Catholic cause. The preachers took alarm at the sudden and unexpected increase of Popery. "Before this French court came to Scotland," said Walter Belcanqual in one of his sermons in 1580 "there were either few or none that durst avow themselves Papists, neither yet publicly in the country, neither in the reformed cities, neither in the king's palace. But since that time, not only begin the Papists within the realm to lift up their heads, but also our Scottish Papists that were outside the realm swarm home from all places like locusts, and have taken such hardihood unto them that not only have they access to the French court, but also in the king's palace, in the particular sessions of our kirks, and general assemblies thereof, durst plainly avow their Papistry, and impugn the truth, both against the laws of the realm and discipline of the Church, contrary to all practice that we have had before."[36]

The members of the General Assembly, annoyed at the attempt of the king to support the episcopal system of government, were determined to remove Lennox, whom they regarded as an emissary of Rome. Elizabeth's agents, too, were busy stirring up discontent. A plot formed by Ruthven Earl of Gowrie, the Earl of Mar, and others, for the capture of the king, was carried out successfully during a visit paid by James to Ruthven's castle at Gowrie (The Gowrie Plot). He was seized and lodged safely in Stirling. The Earl of Arran who attempted to rescue his sovereign was made prisoner, and Lennox was obliged to flee to France (1582).

For a time Melville and the preachers, who gloried in Gowrie's successful machinations, held the king in bondage. The General Assembly of 1582 expressed its approval of what had been done,[37] and renewed its attacks upon the episcopal system. James, however, succeeded in making his escape from confinement; the Earl of Arran was recalled to court; Ruthven was declared a traitor and was beheaded, and the other conspirators were obliged to make their escape to England. James entered into close correspondence with some of the Catholic powers abroad, and even went so far as to appeal to the Pope for assistance against the enemies who surrounded him (1584). For a time it seemed as if a great Catholic reaction was about to set in. Priests who had escaped from England were labouring with success in the Scottish mission-fields; a few Jesuits had arrived from the Continent, and France, Spain, and the Pope were in correspondence regarding the assistance that might be given to James and his mother. But the spies of Elizabeth soon obtained knowledge of what was in contemplation. France and Spain were too jealous of one another to undertake an armed expedition, without which success was impossible. Negotiations were opened up with a view of detaching James from the Catholic party, and of inspiring him with distrust for his mother. As he was always more anxious to secure his accession to the English throne than to defend either his mother's life or her religion, he succumbed completely to English influence.

Not even the execution of his mother in 1587 was sufficient to rouse him to take serious action. Though he was urged by many of the Scottish nobles to declare war he contented himself with angry speeches and protests that passed unheeded. Even many of the Presbyterian lords were ready to support him had he declared war, and Catholic noblemen like the Earls of Huntly, Erroll, and Crawford, Lord Maxwell, and Lord Hamilton, offered their assistance. It was well- known, too, that Philip II. was preparing at the time for an invasion of England. Had Scotland declared war the results might have been disastrous for England, but James, instead of taking the offensive, accepted a pension from Elizabeth and offered to assist in the defence of the kingdom. He endeavoured at first to conciliate the Catholic party by restoring John Leslie Bishop of Ross, who had been for years a most zealous defender of Mary Queen of Scots, to his See and his possessions, and by appointing the exiled Archbishop of Glasgow to be his ambassador at the French court. The General Assemblies, however, backed up by Elizabeth forced him to take strong measures against the adherents of the old religion. In 1593 a proclamation was issued ordering all Jesuits and seminary priests to leave Edinburgh within two hours under pain of death, and a violent campaign was begun in nearly every part of Scotland against the Catholic nobles and clergy. The Catholic lords who were in close communication with Spain were forced to take up arms. Their forces were mustered under the Earls of Huntly and Erroll, and gained a complete victory at Glenlivet over the Earl of Argyll who was dispatched against them. When the news of this defeat reached the king at Dundee he displayed unwonted activity. He assembled a large army to punish his rebellious subjects, and the Catholic lords were at last forced to make their escape from the country. With the flight of Huntly and Erroll (1595) and the dispersal of their troops the triumph of Protestantism in Scotland was assured.