The Jesuits came almost immediately into conflict with the interests of the Spanish colonists. They were welcomed at first, because they were expected to lend themselves to the enslavement of the Indians. When their real purposes were discovered feeling against them rose high. The Creoles clearly saw that it was going to be far more difficult to extend their power over the Indians gathered together in villages under Jesuit protection than over unorganised and friendless bands of unconverted savages.

Before 1610 the number of Jesuits that had come to Paraguay was very small. Among the first was the Father named Thomas Fields, a Scotchman. As a matter of fact, the Jesuits were recruited from all the nations of Europe and under their military system had to go wherever they might be sent. English, Irish, and German names, as well as Spanish, are to be found in the lists of Jesuits who laboured in Paraguay.

In 1608 Philip III. of Spain attended to the complaints that came to him through the powerful chiefs of the Order of the indifference and opposition shown by the settlers and colonial authorities, and gave his royal and official sanction to the Jesuit conversion of the Indians along the Upper Paraná. By this time the Fathers had penetrated across to the Paraná and had followed up that stream far north of the Grand Cataract in latitude 24°, which marks the northern boundary of Paraguay proper. It is hard to understand how they overcame the difficulties of travelling. To this day it is well-nigh impossible to reach the Grand Cataract, and years pass without that wonder of nature's being seen by the eyes of civilised man. No part of the world, outside the Arctic regions, is less accessible than the Paraná above the Grand Cataract. Yet these heroic priests made that region the principal theatre of their operations in the early years of the seventeenth century. The territory is now all Brazilian,—the boundaries of that republic extend on the east bank of the Paraná south nearly to the twenty-sixth degree and on the west bank to the twenty-fourth. The rivers Paranapanema and Ivahy are great tributaries coming down from the east between the twenty-second and twenty-third degrees, and draining a vast extent of the plateau that extends to the Brazilian coast mountains between Curitiba and São Paulo, and on their banks the Jesuits established their principal missions.

In those days there were no clearly defined boundaries between the Portuguese and Spanish dominions. From 1580 to 1640 the king of Spain was also monarch of Portugal. The Jesuits held his royal letters patent for the conversion of the Indians of the province of Guayrá—the name which this remote region bore. They had no reason to anticipate that they would be accused of being invaders of Portuguese territory, or that they would be interfered with by any Portuguese subjects of the Spanish Crown. The nearest Portuguese settlement was at São Paulo, from which Guayrá could be reached only by the long and tedious descent of the Tieté River to its confluence with the Paraná, and thence down that river to the Ivahy. Months would be necessary to make such a journey, great difficulties encountered with waterfalls and rapids, and great privations from want of food in the vast uninhabited regions on the route.

The first Jesuits to arrive after the granting of formal authorisation by the Spanish king were two Italians. They left Asuncion October 10, 1609, and it took them five months of incessant travelling to reach the Paranapanema. The work already done there by the earlier Fathers had borne some fruit. The Indians were prepared for the coming of the new missionaries and readily gathered into the towns which they founded in rapid succession. For the first few years all went well, and within a very short time they claimed to have at least forty thousand souls under their guidance. In 1614 there were 119 Jesuits in Paraguay and Guayrá, and the work of evangelising and reducing to obedience the whole Guarany population of the Paraná valley went on apace. For twenty years these Guayrá missions spread and prospered, while to the east and south the Jesuits acquired more and more influence with the Indians in Paraguay proper, and more and more hemmed in the Creoles of Asuncion.

In 1629 a thunderbolt burst upon Guayrá out of a clear sky. The Portuguese from São Paulo appeared before the Mission of San Antonio and destroyed it utterly, burning the church and houses and driving off the Indians as slaves. Other missions shortly suffered the same fate, and within the short space of three years the towns had been sacked, most of the inhabitants of the region carried off or killed, and the remnants had fled down the river under the leadership of the Fathers. The Paulistas were animated by motives, some good, some bad. Primarily they wished to capture slaves. They hated the Jesuits and had themselves suffered from the latter's system of segregating the aborigines. Only a few decades before, their fathers had destroyed the Jesuit missions near São Paulo, and they were determined not to permit themselves to be hemmed in and crowded out by Indians ruled and protected by Jesuits. They believed in the doctrine of "Brazil for the White Brazilians," and they regarded the Jesuits and their neophytes as natural enemies and fair prey. The sentiment of nationality also animated them. As descendants of Portuguese they hated the Spaniards and their rule. Their allegiance to the Spanish dynasty that had usurped the crown of Portugal sat lightly. The Jesuits came by way of Asuncion, their communications were with the Spanish authorities, and most of them were Spaniards. The Paulistas, as Portuguese, viewed with alarm a rapid spread of Spanish ecclesiastics up the Paraná valley, which threatened soon to reach their own neighbourhood. Avarice, love of adventure, race pride, patriotism, hatred of priestly domination, all co-operated to push them on to undertaking these memorable expeditions.

The great extension of the Jesuits over the northern and eastern regions of the Paraná valley occurred during the period when Hernandarias was the dominant figure of the Plate. Creole though he was, this remarkable man was a friend to the Indian and to the missionary work of the Jesuits. His aid and encouragement in 1609 were essential to the latter's success, for he might easily have nullified the effect of the royal permission to evangelise Guayrá, a formal document that would have been of little value against the delays and excuses of an unwilling governor aided by the jealous people. After his first term as governor at Buenos Aires, the Spanish government determined to put a stop to the more flagrant of the abuses practised against the savages and created the office of "Protector of the Indians." Hernandarias was named to fill it, and carried out his instructions in a moderate spirit. He understood the country and the situation of the colony well, and did not undertake to abolish Indian slavery. In that tropical climate the whites will not labour in the fields so long as there are Indians who can be forced to work, and the Spaniards still regarded the Indian as little better than an animal.

On the other hand, Hernandarias was too intelligent not to see that there must be restraints on the cruelties and exactions of the Creoles if the Indians of Paraguay were to be saved from the extermination that had been the fate of the Haytians a century before. The outcome was, that though a new code of laws was promulgated by the impracticable Spanish king, which forbade any further enslavement of the aborigines, its provisions were largely disregarded. At the same time, however, the Indians acquired a legal status, and their condition was gradually improved until it became not much worse than that of the contemporaneous European peasantry. The Jesuits were guaranteed against interference and allowed to go out into the remoter wilderness and give to the yet unslaved inhabitants the invaluable protection of membership in their missions.

In 1619 the natural and commercial division between Paraguay proper and the rest of the province was officially recognised. The region between the Paraguay and the Paraná rivers was made a separate province, directly dependent upon the Viceroy at Lima and the Audiencia at Charcas in Bolivia. It included officially the Jesuit missions south-east of the Paraná as well as the present territory of Paraguay.

When the Paulistas began their terrible attacks on the Guayrá missions in 1629, the governor of Paraguay refused to send any assistance to the Jesuits. The latter charged him with a corrupt understanding with the invaders, by which he was to share in the profits of the slaves sold. The Order had agreed with the Spanish government not to put any arms into the hands of the Indians, so the latter were defenceless against the Paulistas, who attacked musket in hand. The Creoles and Spaniards in Asuncion resented more and more the presence and power of the Jesuits, and viewed with ill-concealed satisfaction the misfortunes that now overwhelmed the priests. The governor, in declining to send help, was only carrying out the wishes of the people around him. Had the number of whites in Paraguay not been so very small the Jesuits might have been expelled as they were in São Paulo.