Syntaxin-1 is a component of the synaptic vesicle docking and/or fusion soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) complex (7S and 20S complexes) in nerve terminals. Syntaxin-1 also forms a heterodimer with Munc18/n-Sec1/rbSec1 in a complex that is distinct from the 7S and 20S complexes. In this report, we identify a novel syntaxin-1–binding protein, tomosyn, that is capable of dissociating Munc18 from syntaxin-1 and forming a novel 10S complex with syntaxin-1, synaptosome-associated protein of 25 kDa (SNAP-25), and synaptotagmin. The 130 kDa isoform of tomosyn is specifically expressed in brain, where its distribution partly overlaps with that of syntaxin-1 in nerve terminals. High level expression of either syntaxin-1 or tomosyn results in a specific reduction in Ca²⁺-dependent exocytosis from PC12 cells. These results suggest that tomosyn is an important component in the neurotransmitter release process where it may stimulate SNARE complex formation.