Ca2+ stimulates exocytosis in permeabilized insulin-secreting cells. To investigate the putative cytosolic components involved in the Ca2+ response, HIT-T15 cells (a pancreatic B-cell line) were permeabilized with streptolysin-O, a procedure that allows rapid exchange of soluble components including macromolecules. We found that in this cell preparation the secretory response to Ca2+ but not to guanosine 5'-[gamma-thio]triphosphate was lost as a function of time and could be restored by rat brain cytosol in a concentration-dependent manner. Reconstitutive activity of rat brain cytosol was found in a high-molecular-mass heat-labile partially N-ethylmaleimide(NEM)-sensitive fraction. The NEM-sensitive factor (NSF) and the soluble NSF attachment protein (alpha-SNAP) were found to be expressed in HIT-T15 cells and largely lost (about 30% remaining) from porated cells. Recombinant alpha-SNAP partially reconstituted the Ca2+ response when added to the permeabilized cells. Moreover, alpha-SNAP restored the effect of NEM-treated cytosol to the level observed for untreated cytosol. In contrast, NSF was ineffective when preincubated alone or with NEM-treated cytosol. Our results indicate that both alpha-SNAP and NEM-insensitive cytosolic factors are involved in Ca2+-mediated exocytosis from endocrine HIT-T15 cells.