Annexins are a family of calcium-binding proteins that have been implicated in a wide range of intracellular processes. We have previously reported that stimulation of platelets with agents that increase intracellular [Ca# + ] induces the relocation of annexin V to membranes, and that this annexin V may be binding to a 50 kDa protein located within platelet membranes. We report here, using an in itro reconstitution system, that the relocation of annexin V to membranes is enhanced by ATP. We also demonstrate that when adenosine 5h-[γ-thio]-triphosphate, which can replace ATP in phosphorylation reactions, is substituted for ATP, the amount of annexin V that binds to membranes is further increased. In separate experiments using intact cells, we show that the protein phosphatase inhibitor okadaic acid mimics the action