Synaptotagmin I is a Ca2+/phospholipid binding protein of synaptic vesicles with a proposed function as a Ca2+ sensor in synaptic vesicle exocytosis. Using controlled partial proteolysis as an assay, we now show that synaptotagmin I undergoes a conformational change as a function of Ca2+ binding. As observed for phospholipid binding, Ba2+ and Sr2+ but not Mg2+, substitute for Ca2+ in effecting this conformational change. The first C2 domain from synaptotagmin I that represents the Ca(2+)-dependent phospholipid binding domain of synaptotagmin also undergoes a Ca(2+)-dependent change in controlled partial proteolysis. In contrast, no effect of Ca2+ was observed with mutant C2 domains containing point mutations that abolish Ca2+ binding. The Ca2+ concentration dependence of the effect of Ca2+ on proteolysis mirrors the Ca2+ dependence of phospholipid binding. The conformational shift in synaptotagmin I caused by Ca2+/phospholipid binding could be the basis for its Ca(2+)-regulated function in triggering neurotransmitter release.