In a large-scale analysis of gene expression in pancreatic cancer, we isolated the homologue of the mouse Rab20. The mouse protein was previously identified during a search for novel Rab proteins, a family of small GTP-binding proteins involved in the regulation of intracellular vesicular transport. The Rab20 protein has no close relationship to any member of the Rab protein subfamily. In contrast to other members, it contains an insertion of 40 amino acids of unknown function and an inversion of 3 amino acids at the position corresponding to codon 61 in p21ras proteins. Using immunofluorescence and immunoelectron microscopy, we localized the Rab20 protein in the vicinity of the Golgi apparatus. Rab20 expression was detected by Western blot analysis in 11 of 11 pancreatic tumor cell lines and 7 of 8 primary pancreatic carcinomas. Absent or very faint expression was observed in normal pancreas cell extracts. Immunohistochemical analysis of Rab20 in tissues showed low or absent expression in normal pancreas and stronger expression in 15 of 18 exocrine pancreatic adenocarcinomas. Rab20 was also detected in preneoplastic pancreatic intraductal neoplasia lesions, suggesting that its up-regulation may be an early event in pancreatic carcinogenesis.