There has been controversy about the dating of the Missa Papae Marcelli. Pope Marcellus, who had expressed an intention to reform church music so that the words could easily be heard and understood, reigned only for three weeks in early 1555. If the Mass was written during his pontificate, then it must be dated to 1555. If it was in memory of Pope Marcellus or simply a tribute in accordance with his principles, it could have been written at a later date. After the Council of Trent a commission of cardinals was established, meeting in Rome in 1564 and 1565 to consider the question of church music. Legend has it that, as in Pfitzner's opera Palestrina, the composer's work was heard, with others, as an example of what could be done to provide intelligibility in a polyphonic context. The work was, in any case, published in 1567 in Palestrina's second collection of Mass settings. It follows the general guidance that was eventually to result from the Council of Trent in its general clarity of texture and apparent avoidance of a secular cantus firmus, although some have chosen to find in it a subversive reference to the popular L'homme armé (The Armed Man), suggested at the outset and recurring in later movements. In the end, however, the Council did not specifically condemn the use of a secular cantus firmus, although there had been a general injunction against music that was 'lascivious or impure'. Palestrina himself later published two Masses using L'homme armé as a basis, but in general made relatively little use of secular melodies in his music. Polyphonic structures in music of the period, it should be added, were often built on an existing melody, taken in sections and treated imitatively by voice after voice. While this so-called cantus firmus was often derived from plainchant, it could equally well be drawn from secular compositions of one sort or another. While the Council of Trent may have limited musical excesses, it certainly did not put an end to the use of secular sources of material.

The Missa Papae Marcelli is for the most part for six voices, entering in close imitation in the opening Kyrie eleison. The Christe eleison brings an immediate element of homophonic writing at the outset, as pairs of voices answer each other before a fuller texture is explored, and the final Kyrie allows the voices to enter in imitation. The Gloria is in general syllabic and often homophonic in its treatment of the text, as is the Credo, in which the initial cantus firmus is at first heard from the first bass. Palestrina continues to explore the possibilities of contrasted groupings of voices, using a quartet for the words Crucifixus etiam pro nobis, before the six-voice Et in Spiritum Sanctum. The Sanctus finds a place for melismata, a number of notes allocated to one syllable, while the Benedictus is given to four voices. There is a full polyphonic texture in the Agnus Dei, with an element of canonic writing in the second Agnus.