Gene therapy may provide new treatments for severe pancreatic disorders. However, gene transfer to the pancreas is difficult because of its anatomic location and structure, and pancreatitis is a serious concern. Like the human pancreas, the canine pancreas is compact, with similar vascularization and lobular structure. It is therefore a suitable model in which to assess gene transfer strategies. Here we examined the ability of adenoviral vectors to transfer genes into the pancreas of dogs in which pancreatic circulation had been clamped. Adenoviruses carrying the beta-galactosidase (beta-gal) gene were injected into the pancreatic-duodenal vein and the clamp was released 10 min later. These dogs showed beta-gal-positive cells throughout the pancreas, with no evidence of pancreatic damage. beta-Gal was expressed mainly in acinar cells, but also in ducts and islets. Moreover, transduction was prominent in connective tissue of the lobe septa. beta-Gal expression in the exocrine pancreas of a diabetic dog was also found to be similar to that observed in healthy dogs. Thus, efficient gene transfer to canine pancreas in vivo may be achieved by adenovirus injection after clamping pancreatic circulation. This technique may be used to assay new gene therapy approaches for diabetes mellitus and other pancreatic disorders.