We examined the action of mastoparan on beta cell exocytosis. Mastoparan stimulated GABA and insulin release from MIN6 beta cells. On the other hand, mastopraran-induced GABA release was decreased by expressing the tetanus toxin C1 light chain in MIN6 cells. We have then investigated the relationship between SNARE proteins and mastoparan action using adenovirus-mediated gene transfer system. Overexpression of t-SNAREs, syntaxin 1A, and SNAP-25 inhibited the mastoparan-induced insulin release by approximately half-fold of control levels, however, the mastoparan-induced GABA release was not affected by these t-SNAREs overexpression. The overexpression of mutant alpha-SNAP (1-285), which inhibits the wild-type alpha-SNAP function in a dominant negative manner, did not influence either mastoparan-induced GABA or insulin release in spite of its marked inhibition of glucose-stimulated insulin release. Our data indicate that mastoparan stimulates GABA exocytosis via vesicular transport; however, SNARE proteins are differently involved in the exocytosis of insulin and GABA.