Rab3 proteins are members of the family of Ras-like monomeric GTP-binding proteins that have been implicated in secretion in neuronal cells. Although an isoform of Rab3 has been assumed to exist in pancreatic acini, its identity has not yet been established. We now report that Rab3D is present in rat pancreatic acini and is localized to the zymogen granule membrane. Reverse transcription-polymerase chain reaction (PCR) was used with primers based on mouse Rab3D to amplify Rab3D from rat pancreas. The PCR product without primer sites consisted of 580 base pairs and was 94% identical to the mouse Rab3D cDNA sequence previously cloned from adipocytes. Western blotting with a polyclonal antiserum raised against Rab3D-specific carboxyterminal amino acids identified Rab3D in rat pancreatic acini and revealed its concentration on zymogen granule membranes. Immunocytochemistry of pancreatic lobules showed that Rab3D localized to the apical region in a pattern similar to amylase. Confocal fluorescence microscopy of lobules double immunolabeled with antibodies to Rab3D and the granule membrane marker protein glycoprotein-2 (GP-2) revealed a similar localization of these proteins to zymogen granules. Immunocytochemistry also revealed the presence of Rab3D in chief and enterochromaffin-like cells in the stomach, acinar cells in lacrimal and parotid gland, and Paneth cells in the intestine. These results show that Rab3D is expressed in rat pancreatic acini and other exocrine secretory cells. Its location implies it may be involved in regulated exocytosis.