The Immaculate Conception was one of the most important themes of Counterreformation art. The Immaculate Conception does not refer to Christ’s virgin birth, but to the belief that Mary herself was conceived free of original sin. Starting in the sixteenth century, an iconographic model for the representation of the dogma developed in Spain. Two passages in the bible were decisive in its development, one in The Song of Solomon, and the other in the Revelation of St. John the Evangelist.

El Greco takes his inspiration for this painting from the passage in the Apocalypse in particular. In the lower left corner of the painting we can see St. John at the moment he perceives his vision, which is described in the New Testament thus, "and there appeared a great wonder in the heaven; a woman clothed with the sun, and the moon under her feet, and upon her head a crown of twelve stars". El Greco models the saint’s athletic body rather soberly, in a naturalistic, realistic manner, whilst the figure of the Virgin Mary is elongated and imbued with the spiritual and dynamic nature of the vision.