Ca(2+)-dependent neurotransmitter release consists of at least two components: a major fast component that is insensitive to Sr2+ and a minor slow component that is potentiated by Sr2+ (Goda, Y., and Stevens, C. F. (1994) Proc. Natl. Acad. U. S. A. 91, 12942-12946). These results suggest that at least two Ca2+ sensors act in synaptic vesicle fusion with distinct Ca2+ and Sr2+ binding properties. We have now investigated the relative Ca2+ and Sr2+ binding activities of synaptotagmins to evaluate their potential roles as Ca2+ sensors for the fast and slow components. Our results demonstrate that the first C2 domains of synaptotagmins I, II, III, V, and VII have very similar Ca2+ requirements for phospholipid binding (range of EC50 = 2.6 microM to 5.0 microM), but distinct Sr2+ requirements (EC50 range = 23 microM to 133 microM); synaptotagmins I and II had the lowest Sr2+ affinity, and synaptotagmin III the highest Sr2+ affinity. Purified synaptotagmin I from bovine brain exhibited similar properties as its recombinant first C2 domain, suggesting that the first C2 domain fully accounts for its Ca(2+)-dependent phospholipid binding properties. Sr2+ was unable to trigger syntaxin binding by synaptotagmin I at all concentrations tested, whereas it was effective for synaptotagmin III. These results suggest that different C2 domains have distinct Sr2+ binding properties. They support the hypothesis that synaptotagmins localized on the same vesicle perform distinct functions, with synaptotagmins I and II serving as candidate Ca2+ sensors for the fast component in release and synaptotagmin III for the slow component.