Annexin (Anx) VI has been implicated in mediating the endosome aggregation and vesicle fusion in secreting epithelia during exocytosis. In addition, AnxVI of porcine liver is an ATP-binding protein, and ATP in vitro modulates its interaction with membranes and cytoskeletal elements (Bandorowicz-Pikuła and Awasthi, FEBS Lett. 409 (1997) 300-306). In this study, we examined the effect of ATP on phosphatidylserine (PtdSer) aggregation in the presence of annexin and on calcium-dependent binding of protein to liposomes, and found that ATP stimulates the former process, although it increases the calcium concentration necessary for half-maximal binding of AnxVI to membranes. These results were corroborated by the experiments with fluorescent analog of ATP, in which binding of ATP to AnxVI was affected by binding of Ca2+ and/or phospholipids to the protein. Taken together they favour an idea of ATP being a functional ligand for AnxVI, which even in the relative absence of Ca2+ may modulate interaction of AnxVI with PtdSer-enriched membranes.