Background and Aims: Pancreatic stem cells are powerful tools for future gene/cell therapy for diabetes, pancreatic carcinoma and chronic pancreatitis. However, it is unclear where the pancreatic stem cells exist in adult pancreas. To identify the pancreatic stem cells, we have focused on a homeodomain-containing transcription factor, pancreatic/duodenal homeobox-1 (PDX-1), and an intermediate filament protein, nestin, which are important candidates of pancreatic stem cell markers. Methods and results: We investigated the immunohistological localization for PDX-1 and nestin in adult rat pancreas. PDX-1 staining was detected in small evaginations of the main pancreatic duct and in nuclei of islet cells. Nestin staining was also detected in small evaginations of the main duct, islets and spindle-shaped cells in the connective tissue around the main duct. These spindle-shaped cells were also positive for -smooth muscle actin, the marker of myofibroblasts. Confocal laser microscopy study showed that nestin and PDX-1 are co-localized not only in a subset of cells in small evaginations of the main duct but also in a few cells in the islets. These cells of the main duct were also positive for the ductal cell marker, cytokeratin 19. Conclusion: Our results suggest that the double positive cells for PDX-1 and nestin might be important candidates for pancreatic stem cells in adult rats.