The site for the catacombs of St.Sebastian had been originally used as a quarry for pozzolana, or soft volcanic rock.   It was common practise in ancient times for former quarries to become burial grounds for slaves and freedmen, a place for modest graves, usually no more than simple loculi or columbarium niches.
Several grand mausolea, however, were also built in this graveyard, collected together in an area known as the "piazzuola", a relatively large open space that today is completely buried underground.

Three richly decorated mausolea were built in this space, probably for wealthy freedmen and their families. The monumental facades all resemble each other, all with the door at center surmounted by an inscription naming the proprietor, a tympanum decorated with pictures and attic where probably the commemorative celebrations were held for the deceased, including the annual memorial banquet held on the anniversary of his or her death. The first mausoleum constructed on the site was that of Marcus Clodius Hermes. The room on the ground floor of the Hermes mausoleum has both arcosolia and loculi for burial. Below this level is another room with more arcosolia. The second mausoleum on the piazzuola is called that of the "Innocentiores", perhaps to indicate the name of an association. In this case, there are no rooms on the ground floor of the mausoleum, but a steep stairway which leads to a subterranean burial chamber. The stucco reliefs which cover the vault of the crypt are particularly noteworthy, including representations of a shell and peacock. The third mausoleum, the last, is known by the image of an axe in stucco on its facade. This, too, was built with two floors for burial in arcosolia and loculi. In the tufa walls which form the outer perimeter of the piazzuola many other loculi were created, destined, most scholars believe, for pagan burials although in a later phase of burial activity in the piazzuola, several Christians were also interred there, as is gathered from the representations of the fish and anchor found which are typical Christian symbols. Around the middle of the third century, the topography of the site was drastically altered when the piazzuola was completely buried under a landfill and a large space was created on this new upper level.

This phase was characterized by the creation of a triclia, a covered space accessed from a small stairway, with the features of an colonnaded hall furnished with benches attached to its back wall. Other than the triclia, the space held also a small apsidal monument with a shrine and niche inside. Although there is no definite proof as to the shrine's significance, many scholars believe that it was created for, and for a time held, the remains of the greatest of the apostles and martyrs, Peter and Paul. The alterations on the site may have been undertaken precisely for this purpose. In addition to the ancient sources which record the veneration paid to the two apostles at this site, the "Memorial to the Apostles", numerous scratchings in the plaster of the back wall of the triclia, to all effects ancient graffiti, have been found to be invocations and prayers made by the faithful to Peter and Paul. These testimonials strongly suggest that there was an active cult of the two saints in the area. Several examples are still legible and are interesting for what they reveal; for example, the petition of a certain Victor, who at some point during the third century wrote "Paul and Peter, pray for Victor", requesting the intercession from two of the most celebrated martyrs for the Christian faith. The remains of the two saints would have been translated to the site of the Saint Sebastian catacombs by the year A.D. 256, out of fear that during the persecution of the Emperor Valerian the apostles's tombs would be violated and their remains destroyed.

Only in a later and more secure period could they be returned to the cemeteries on the via Ostiense and Vatican hill. Some doubts about these events remain, however, for although the evidence for a cult of Peter and Paul is strong at the Saint Sebastian site, there may never have been in fact the actual remains of the apostles at that particular cult shrine. The evidence continues to be hotly debated in scholarly circles. It must also be noted that during the period when the cult of the apostles was active on the site, the area was not in use for new burials and therefore would have been used only for commemorations of the martyrs enshrined there. Finally, during the fourth century, the shrine and hall were in turn buried under the foundation of the new Constantinian basilica "of the apostles".