The membrane proteins synaptobrevin, syntaxin, and SNAP-25 form the core of a ubiquitous fusion machine that interacts with the soluble proteins NSF and alpha-SNAP. During regulated exocytosis, membrane fusion is usually strictly controlled by Ca2+ ions. However, the mechanism by which Ca2+ regulates exocytosis is still unclear. Here we show that the membranes of exocrine secretory granules contain an 18-kDa protein, syncollin, that binds to syntaxin at low Ca2+ concentrations and dissociates at concentrations known to stimulate exocytosis. Syncollin has a single hydrophobic domain at its N-terminus and shows no significant homology with any known protein. Recombinant syncollin inhibits fusion in vitro between zymogen granules and pancreatic plasma membranes, and its potency falls as Ca2+ concentration rises. We suggest that syncollin acts as a Ca2(+)-sensitive regulator of exocytosis in exocrine tissues.