We have detected high- and small-molecular-weight GTP-binding proteins (G- and smg-proteins, respectively) in zymogen granule membranes (ZGM) of rat pancreatic acinar cells. Gi-proteins with molecular weights of 40/41 kDa were detected by pertussis-toxin induced ADP ribosylation and by [α-32P] GTP photoaffinity labelling in ZGM. Smg-proteins were analysed by [α-32P] GTP binding, ADP ribosylation with Clostridium botulinum ADP ribosyltransferase C3 and immunoblotting following one- and two-dimensional gel electrophoresis. Two-dimensional separation of ZGM proteins revealed the presence of up to 30 differently charged smg-proteins with molecular masses between 18 and 27 kDa in ZGM. A monoclonal antibody, which recognizes both rab3A and rab3B (clone 42.1), identified 2 proteins with isoelectric points (pI) of 4.89 and 4.96 as proteins closely related to rab3. These proteins were mainly present in ZGM and to a slight extent also in plasma membranes (PM), microsomal membranes (MM) and cytosol. An antibody, which recognizes rab3A only (clone 42.2), did not react with pancreatic proteins. An anti-rap IB antibody recognized a 23 kDa protein localized in the ZGM, PM and to a lesser extent in MM. A monoclonal antibody against all 3 known ras proteins (p21Ha-ras, p21ĸi-ras, p21N-ras) identified ras proteins only in PM and not in ZGM or other membranes. C3-induced ADP ribosylation as indication for the presence of rho and/or rac proteins was detected in PM, MM and cytosol but not in ZGM. The presence of multiple GTP-binding proteins in ZGM suggests a possible role of these proteins in the regulation of exocytosis in pancreatic acinar cells.