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At the al-Aqsa mosque compound, the Pope urged people of all religions to "work together for justice and peace".
He then prayed at the Western Wall, which lies just beneath it, bowing his head as he touched the stones.
The pontiff has been feted by Israel and the Palestinians, and has invited their presidents to the Vatican.
Both Israel's Shimon Peres and Palestinian leader Mahmoud Abbas have given a positive response.
'Brothers and sisters'
Pope Francis began the third day of his trip by visiting the compound, considered the third holiest site in Islam.
Known to Muslims as the Haram al-Sharif (Noble Sanctuary) and Jews as the Temple Mount, the status of the site is one of the most contentious issues in the Israel-Palestinian conflict .