When archaeologists discuss ‘ancestor cults’ or ‘ancestor veneration’, what this might entail in practice usually remains vague, leading to charges that the concept of ‘ancestors’ is often applied generically. In this article, the authors combine bioarchaeological, taphonomic, radiocarbon, and isotopic studies to explore the ritual practice of the selective retention, curation, and deposition of a group of human crania and mandibles. Between 5500–5400 bc, Neolithic people at Masseria Candelaro (Puglia, Italy) deposited broken crania and mandibles from about fifteen individuals in a heap in the centre of the village. These individuals were mostly probable males, collected over the course of two centuries and actively used, with their deposition marking the final disposal of a ritual collection. The motivations for the curation of cranial bone are investigated through comparison with archaeological and ethnographic examples, advancing an interpretation of ritual practice directed towards ancestors.