The biosynthesis of GP-2, the major glycoprotein associated with zymogen-granule membranes in the pancreas, was studied in acinar cell suspensions from rat pancreas. Pulse-chase experiments, using [35S]methionine, were performed and the processing of GP-2 was analyzed by immunoprecipitation and sodium dodecyl sulfate/polyacrylamide gel electrophoresis. GP-2 is synthesized as a precursor glycoprotein with apparent molecular weight Mr = 73000. Within 60 min after synthesis it is almost completely converted to the mature form (Mr = 78000-80000). Only the precursor form of GP-2 is sensitive to digestion with the glycosidase endo-beta-N-acetylglucosaminidase H, indicating that the observed conversion reflects the processing of 'high-mannose' oligosaccharides into complex type oligosaccharides. Acinar cells cultured in the presence of increasing concentrations of the N-glycosylation inhibitor tunicamycin synthesize 5-6 distinct precursor GP-2 species with apparent molecular weights decreasing from 73000-61000. We conclude that GP-2 contains five or six N-linked carbohydrate chains. From cell fractionation studies it was established that the precursor GP-2 is present in a microsomal fraction with high density (greater than 1.169 g/ml) presumably derived from the rough endoplasmic reticulum; mature GP-2 is localized in low density microsomes (less than 1.130 g/ml) probably Golgi vesicles. The GP-2 in zymogen granule membranes is also in the mature form.