Synaptic vesicles are proposed to dock at the presynaptic plasma membrane through the interaction of two integral membrane proteins of synaptic vesicles, VAMP and synaptotagmin, and two plasma membrane proteins, syntaxin and SNAP-25. We have characterized the binding properties of these proteins and observed SNAP-25 potentiation of VAMP 2 binding to syntaxins 1a and 4 but not syntaxins 2 or 3. n-sec1, a neuron-specific syntaxin-binding protein, bound syntaxin with nanomolar affinity, forming a complex that is distinct from the previously identified 7S and 20S syntaxin-containing complexes. This suggests that syntaxin exists in at least three states: bound to n-sec1, in a 7S particle, and in a 20S particle. Recombinant n-sec1 inhibited VAMP or SNAP-25 binding to syntaxin. We propose that the specific associations of VAMP, SNAP-25, and syntaxin mediate vesicle docking and that a syntaxin/n-sec1 complex precedes and/or regulates formation of these complexes.