Synaptotagmin, a synaptic vesicle protein involved in Ca(2+)-regulated exocytosis, displayed direct high affinity interaction with neuronal sodium channels. Monoclonal antibodies directed against synaptotagmins I and II adsorbed in a concentration-dependent and -specific manner [(3)H]saxitoxin prelabeled sodium channels extracted with detergent from nerve endings. Conversely, co-immunoprecipitation of synaptotagmin was achieved by antibodies against sodium channel subunits. Consistent with the co-immunoprecipitation assays, solubilized [(3)H]saxitoxin-prelabeled sodium channels were trapped on immobilized maltose binding protein (MBP)-synaptotagmin I. In vitro recombinant protein assays were employed to identify the interaction site of synaptotagmin I, which was located on the cytoplasmic loop between domains I and II of the sodium channel alphaIIA subunit. The co-immunoprecipitated synaptotagmin-sodium channel complexes were found to be Ca(2+)-dependent; this effect was mimicked by Ba(2+) and Sr(2+) but not Mg(2+). Finally the complex was shown to be distinct from the synaptotagmin-SNARE protein complex that can selectively interact with presynaptic calcium channels (N and P/Q types). Thus, our findings demonstrate an unexpected and direct interaction between sodium channels and synaptotagmin. The Ca(2+)-regulated association between sodium channels and a protein implicated in vesicular fusion may have intriguing consequences for the establishment and regulation of neuronal excitability.