The function of soluble N-ethylmaleimide-sensitive attachment protein-alpha (alpha-SNAP) in exocytosis still remains obscure. This study was conducted to determine the physiological role of alpha-SNAP in the secretion of insulin and gamma-aminobutryric acid (GABA) from pancreatic beta cells. Reverse transcriptase-polymerase chain reaction analysis of total RNA isolated from rat islets disclosed alpha-SNAP, but not beta-SNAP, mRNA expression, and an immunofluorescence study of rat pancreas showed that alpha-SNAP was present predominantly in the cytoplasm of the islets of Langerhans. alpha-SNAP overexpression in rat islets enhanced insulin release relative to the control levels. An in vitro binding study showed that both wild-type alpha-SNAP and C-terminal-deleted alpha-SNAP mutant (1-285) can bind to syntaxin 1A. alpha-SNAP mutant (1-285) was overexpressed to evaluate its activity as dominant-negative effector on insulin release. Overexpression of alpha-SNAP mutant (1-285) in rat islets and MIN6 cells decreased glucose-stimulated insulin release to about 50% of the control levels. Suppression of endogeneous alpha-SNAP in MIN6 cells by treatment with an antisense phosphorothioate oligonucleotide resulted in inhibition of insulin release. In order to examine if alpha-SNAP functions in exocytosis from synaptic-like microvesicles in pancreatic beta cells, the functional role of alpha-SNAP in GABA release from MIN6 cells was studied. The data showed no effect of alpha-SNAP mutant (1-285) overexpression on GABA release. We conclude that 1) alpha-SNAP plays a crucial role in insulin exocytosis via large dense core vesicles, but not GABA released via synaptic-like microvesicles, in pancreatic beta cells; and 2) the interaction of alpha-SNAP and syntaxin 1A may play an important role in the insulin exocytotic process.