Stable isotope analysis and AMS dating of human and animal bone from the Bulgarian Black Sea coastal sites of Varna I and Durankulak provide fascinating insights into their spatial development as Neolithic and Aeneolithic (Copper Age/Chalcolithic) cemeteries, their associated chronologies, and the dietary signatures of the individuals buried there. The question of the Black Sea reservoir effect is explored, together with a comparison of dietary signatures with other European populations. The isotopic results suggest the populations of Varna and Durankulak primarily utilised C3, terrestrial-based protein sources, despite their presumed proximity to the Black Sea. No significant correlation was found between the isotopic signatures and the material wealth of individuals at Varna, which may have implications for theories regarding emergent social/economic hierarchies in prehistory. The AMS results suggest the Varna I cemetery was utilised for a short period of 70–155 years (±1 σ ) in the middle of the fifth millennium BC. Although only a small proportion of the Durankulak burials was dated, evidence suggests the cemetery was in use for at least half a millennium and burials had likely ceased by 4450 BC, in the early part of the Late Aeneolithic, at approximately the same time as those at Varna I. The implications of the AMS dates are discussed in relation to the development of a Black Sea innovation centre in ceramics and metallurgy and the diffusion of these innovations to other parts of the Balkans and the north Pontic zone. Critical issues are identified for Early and Middle Aeneolithic chronology with respect to the dating sequence of the Durankulak cemetery.