SV2 (Synaptic Vesicle Protein 2) is expressed in neurons and endocrine cells where it is required for normal calcium-evoked neurosecretion. In mammals, there are three SV2 genes, denoted SV2A, B and C. SV2A interacts with synaptotagmin, the prime candidate for the calcium sensor in exocytosis. Here, we report that all isoforms of native SV2 bind synaptotagmin and that binding is inhibited by calcium, indicating that all isoforms contain a common calcium-inhibited synaptotagmin-binding site. The isolated amino termini of SV2A and SV2C supported an additional interaction with synaptotagmin, and binding at this site was stimulated by calcium. The amino-terminal binding site was mapped to the first 57 amino acids of SV2A, and removal of this domain decreased calcium-mediated inhibition of binding to synaptotagmin, suggesting that it modulates calcium's effect on the SV2-synaptotagmin interaction. Introduction of the amino terminus of SV2A or SV2C into cultured superior cervical ganglion neurons inhibited neurotransmission, whereas the amino terminus of SV2B did not. These observations implicate the SV2-synaptotagmin interaction in regulated exocytosis and suggest that SV2A and SV2C, via their additional synaptotagmin binding site, function differently than SV2B.