The pancreatic zymogen granule membrane protein GP-2 was introduced into cells of exocrine or endocrine origin by transfection of its cDNA in order to investigate the mechanisms by which proteins are specifically incorporated into the membranes of secretory granules. Permanent transformants expressing GP-2 were isolated from exocrine pancreatic-derived AR42J cells as well as AtT20 cells of anterior pituitary origin and insulinoma-derived Rin5F cells. In AR42J cells, GP-2 was localized by immunofluorescence and immunoelectron microscopy to the endogenous zymogen-like granules as well as to the plasma membrane. In experiments supporting the localization data, incubation of the AR42J transformants with the secretagogue cholecystokinin (CCK8) resulted in enhanced release of a shed form of GP-2 into the medium in parallel with amylase, suggesting that the two proteins were secreted from the same compartment. By contrast, when expressed in AtT20 cells, the protein was found by immunofluorescence microscopy on the plasma membrane as well as in intracellular vesicles that differed in size and location from the endogenous secretory vesicles. By electron microscopy, large (approximately 0.5 micron) multivesicular structures were observed. Single- and double-label immunoelectron microscopy demonstrated that these large organelles labeled with anti-GP-2 antibodies, whereas the smaller adrenocorticotropic hormone (ACTH)-containing secretory vesicles did not. In permanent transformants of Rin5F cells, GP-2 was also excluded from the insulin-containing granules and found in multivesicular bodies similar to those in the AtT20 cells and containing the endosomal/lysosomal marker endolyn-78. Despite the apparent accumulation of GP-2 in lysosome-like structures, it turned over slowly and did not undergo rapid endocytosis from the cell surface. We conclude that GP-2 is targeted to secretory granule membranes by cell type-specific mechanisms that likely involve its interaction with other membrane or content proteins expressed only in the exocrine cells.