Syntaxin is a key presynaptic protein that binds to N- and P/Q-type Ca(2+) channels in biochemical studies and affects gating of these Ca(2+) channels in expression systems and in synaptosomes. The present study was aimed at understanding the molecular basis of syntaxin modulation of N-type channel gating. Mutagenesis of either syntaxin 1A or the pore-forming alpha(1B) subunit of N-type Ca(2+) channels was combined with functional assays of N-type channel gating in a Xenopus oocyte coexpression system and in biochemical binding experiments in vitro. Our analysis showed that the transmembrane region of syntaxin and a short region within the H3 helical cytoplasmic domain of syntaxin, containing residues Ala-240 and Val-244, appeared critical for the channel modulation but not for biochemical association with the "synprint site" in the II/III loop of alpha(1B). These results suggest that syntaxin and the alpha(1B) subunit engage in two kinds of interactions: an anchoring interaction via the II/III loop synprint site and a modulatory interaction via another site located elsewhere in the channel sequence. The segment of syntaxin H3 found to be involved in the modulatory interaction would lie hidden within the four-helix structure of the SNARE complex, supporting the hypothesis that syntaxin's ability to regulate N-type Ca(2+) channels would be enabled after SNARE complex disassembly after synaptic vesicle exocytosis.