Membrane fusion resulting in neurotransmitter secretion forms the basis of neural communication. Three multimeric complexes of the protein syntaxin are important in this process: syntaxin and n-sec1; syntaxin, VAMP, and SNAP-25; and syntaxin, VAMP, SNAP-25, alpha SNAP, and NSF (20S complex). In this report, we demonstrate that unique, yet overlapping, domains of syntaxin are required to form these complexes. The formation of higher order heteromultimers has a set of structural requirements distinct from those required for dimeric interactions. Dissociation of the 20S complex by NSF following ATP hydrolysis requires amino-terminal regions of syntaxin that are outside of the binding domains for the 20S constituent proteins. These data are consistent with the hypothesis that conformational changes in syntaxin, resulting from protein-protein interactions and ATP hydrolysis by NSF, mediate neurotransmitter release.