Pancreatic acinar cells, a model cell type for the study of exocytosis in non-excitable cells, were used here to test the hypothesis that molecular mechanisms regulating exocytosis from neuronal and neuroendocrine cells may also be utilized in exocrine cells. Using specific antisera raised against vesicle-associated membrane protein (VAMP) isoforms 1 and 2, we have identified VAMP-2, but not VAMP-1, immunoreactive proteins on rat pancreatic acinar cell secretory (zymogen) granules. This 18-kDa protein co-migrated with rat brain synaptic vesicle VAMP-2. Tetanus toxin (TeTx) light chain caused complete cleavage of this protein, which was prevented by the addition of EDTA. This toxin also inhibited in a dose-dependent manner Ca(2+)-stimulated enzyme secretion by up to approximately 30% in streptolysin O-permeabilized acini. This effect of TeTx on secretion was prevented by the zinc endopeptidase inhibitor captopril or by boiling the toxin. Incomplete inhibition of secretion by TeTx may suggest that TeTx-insensitive or VAMP-2-independent mechanisms also regulate exocytosis. In support, TeTx light chain was without effect on Rab3AL (effector domain peptide of Rab3)-mediated potentiation of Ca(2+)-stimulated secretion. These results indicate that a TeTx-sensitive VAMP-2-like protein on zymogen granules participates in Ca(2+)-regulated pancreatic enzyme secretion and that undefined Rab3AL-activated mechanisms may act independently to regulate exocytosis.