unc-13 mutants in Caenorhabditis elegans are characterized by a severe deficit in neurotransmitter release. Their phenotype is similar to that of the C. elegans unc-18 mutation, which is thought to affect synaptic vesicle docking to the active zone. This suggests a crucial role for the unc-13 gene product in the mediation or regulation of synaptic vesicle exocytosis. Munc13-1 is one of three closely related rat homologues of unc-13. Based on the high degree of similarity between unc-13 and Munc13 proteins, it is thought that their essential function has been conserved from C. elegans to mammals. Munc13-1 is a brain-specific peripheral membrane protein with multiple regulatory domains that may mediate diacylglycerol, phospholipid, and calcium binding. In the present study, we demonstrate by three independent methods that the C terminus of Munc13-1 interacts directly with a putative coiled coil domain in the N-terminal part of syntaxin. Syntaxin is a component of the exocytotic synaptic core complex, a heterotrimeric protein complex with an essential role in transmitter release. Through this interaction, Munc13-1 binds to a subpopulation of the exocytotic core complex containing synaptobrevin, SNAP25 (synaptosomal-associated protein of 25 kDa), and syntaxin, but to no other tested syntaxin-interacting or core complex-interacting protein. The site of interaction in syntaxin is similar to the binding site for the unc-18 homologue Munc18, but different from that of all other known syntaxin interactors. These data indicate that unc-13-related proteins may indeed be involved in the mediation or regulation of synaptic vesicle exocytosis by modulating or regulating core complex formation. The similarity between the unc-13 and unc-18 phenotypes is paralleled by the coincidence of the binding sites for Munc13-1 and Munc18 in syntaxin. It is possible that the phenotype of unc-13 and unc-18 mutations is caused by the inability of the respective mutated gene products to bind to syntaxin.