Night-time temperatures there have dropped below freezing recently.
Around 30 people, Italians and foreigners, have accepted the invitation to sleep inside St Calixtus church, whose foundations were laid near a well where Pope Calixtus I was martyred in 222, the Vatican said.
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Inside the church, which is Vatican property, the homeless are using sleeping bags or blankets.Since the stone interior of the church is cold itself, electric heaters have been placed on the floor.
Pope Francis has stressed helping those on society's margins.
The Vatican has also let homeless people get warm in some of its vehicles parked near St Peter's Square at night.