Zymogen granules of the exocrine pancreas are the secretory organelles responsible for the regulated secretion of digestive enzymes. Several proteins are associated with or are integral components of the lipid bilayer that forms the zymogen granule membrane. These proteins likely represent important components in the regulated secretion of digestive enzymes. VAMPs (vesicle-associated membrane proteins)/synaptobrevins are a family of 18-kDa integral membrane proteins originally characterized in synaptic vesicles. Polyclonal antisera raised against either a VAMP/glutathione S-transferase (GST) fusion protein or rat brain synaptic vesicles, detected an 18-kDa immunoreactive protein in zymogen granule membranes that co-migrates electrophorectically with rat brain synaptic vesicle VAMP. Rat brain synaptic vesicle VAMP was detected by both antisera. Botulinum-B toxin treatment of zymogen granule membranes did not result in cleavage of zymogen granule membrane VAMP, indicating that exocrine pancreatic VAMP is either VAMP1 or a novel VAMP-isoform. Immunofluorescent studies demonstrated that exocrine pancreatic VAMP localized with GP2, a zymogen granule membrane protein, to the apical region of pancreatic acinar cells. No significant labeling was observed in basolateral regions of pancreatic acinar cells. These results establish the presence of a VAMP protein in the zymogen granule of the rat pancreas and suggest that VAMPs have a role in exocrine secretion.