The Holy Trinity (the Father, the Son and the Holy Ghost) presides over the composition. The Virgin Mary and Saint John the Baptist appear on the left, and further down are characters from the Old Testament, each identifiable by his attributes. The female figure with her back to the viewer has been identified as the Eritrean Sibyl or as Mary Magdalene. On the right, angels accompany members of the Imperial family wearing shrouds and expressions of supplication. They are led by Carlos V (1500-1558). The two old men at the bottom have been identified as Titian and Aretino.

There have been different interpretations of this painting that was conceived as a visual rendering of the Hapsburg's Trinitarian orthodoxy. The work acquired a devotional nuance when Carlos V asked to see it before dying. It is based on a passage by Saint Augustine in which he narrates a heavenly vision of the blessed. The composition is said to draw on Albrecht Dürer and Lorenzo Lotto.

Signed on a paper being held by Saint John the Evangelist, this painting hung in the Aula de Moral at El Escorial until it entered the Prado Museum in 1837.