Fray Luis was arrested by an officer of the Spanish Inquisition on the evening of 27 March 1572. He cannot have been unprepared. His friend and colleague Gaspar de Grajal, who had beaten him in the contest for the Chair of Bible in 1560, had been arrested at the beginning of the month, and Martinez de Cantalapiedra, the Professor of Hebrew, the previous day. Between December 1571 and March 1572 a procession of accusers denounced the three Hebraists to the Valladolid Inquisitors, and more denunciations poured in after their arrests.

On 2 December 1571, seventeen propositions allegedly held by the three were presented to the Supreme Council of the Holy Office by Pedro Fernández, the Dominican Prior of Salamanca. Another leading Dominican, Bartolomá de Medina, testified before the Inquisitors on 17 December and 18 February. He told them that in the University there was too much love of ‘innovations’ (novedades) and not enough of the old ways of religion and faith. He accused the three professors of fomenting this: ‘Prefieren a Vatablo, Pagnino y sus judíos, a la traslación Vulgata y al sentido de los Santos’ (x, 7), ‘They prefer Vatable, Pagnini and their Jews to the Vulgate translation and the sense of the Saints’. On 26 December a student denounced Fray Luis's translation of the Song of Songs, copies of which were being circulated.