Synaptotagmin 1 (SytI) is a synaptic vesicle protein that binds Ca2+ and is essential for fast, Ca(2+)-dependent neurotransmitter release in the hippocampus, suggesting that it serves as a Ca2+ sensor for exocytosis. Although SytI has two cytoplasmic C2-domains, only the first C2-domain was shown to exhibit Ca2+ regulation; it binds phospholipids and syntaxin in a Ca(2+)-dependent manner. By contrast, the second C2-domain is inactive in these assays and only binds putative interacting molecules in a Ca(2+)-independent manner. We have now discovered in a yeast two-hybrid screen for SytI-interacting molecules that the C2-domains of SytI interact with themselves. Using immobilized recombinant C2-domains from SytI and SytII, we found that only the second but not the first C2-domains of these synaptotagmins are capable of affinity-purifying native rat brain SytI and that this binding is Ca(2+)-dependent, suggesting that only the second C2-domain is capable of a Ca(2+)-triggered self-association. A relatively high Ca2+ concentration (> 100 microM) is required for binding in the presence of Mg2+; Sr2+ and Ba2+ but not Mg2+ can substitute for Ca2+. Our data suggest that the second C2-domain of SytI is also a Ca(2+)-regulated domain similar to the first C2-domain but with distinct binding activities.