The pope has donned his iconic hat for the occasion of the mass, an intimate affair that has the potential to become a flashpoint of Vatican-Jewish tensions.
The room is not often used for Christian rites, in a bid by Israel to keep the delicate status quo of the site.
The church is talks with Israel for increased rights to hold services there, but some ultra-Orthodox and Jewish nationalists have lobbied against any changes to the site, which is also said to hold the tomb of King David.
Israeli and Vatican officials have insisted that talks are not over ceding the building to the church, but rumors to that effect have persisted, further exacerbating tensions.
Ahead of the pope’s visit, police issued orders against a number of Jewish ultra-nationalists to stay away from parts of Jerusalem, in a bid to keep them from disturbing the pope’s visit.