In this study we have determined which Galpha proteins are associated with the pancreatic zymogen granule membrane. The purity of the granule preparation was evaluated both morphologically and biochemically. These studies demonstrated that the isolated zymogen granules were free of any significant cross contamination from other organelles including the plasma membrane. Western blot analysis showed that Goalpha, Gqalpha, and Gsalpha are associated with the zymogen granule membrane, but that Gi1alpha, Gi2alpha, and Gi3alpha are not. The location of these Galpha proteins suggests that they may modulate regulated exocytosis in the pancreatic acinar cell.