We examined whether retrograde intrabiliary adenoviral administration could induce safe and efficient transgene expression in hepatocytes. We administered recombinant adenovirus carrying a reporter lacZ gene retrogradely into the common bile duct of rats and evaluated the transduction efficiency of the lacZ gene in the liver histochemically by X-gal staining, and also quantitatively by a chemiluminescent reporter gene assay. Retrograde administration of adenovirus into the common bile duct was shown to successfully induce transgene expression in the liver. Although transgene expression induced by intrabiliary adenoviral administration was observed predominantly at periportal areas, a considerable number of cells expressing the transgene were detectable even in lobular and centrilobular areas. Furthermore, histochemical analysis revealed that intrabiliary adenoviral administration resulted in gene transfer into hepatocytes, but not into biliary epithelial cells. Transgene expression in the liver was transient, and pathological and biochemical analyses revealed that hepatic damage caused by intrabiliary adenoviral administration was not substantial. The results demonstrated in the present study suggest that retrograde administration of adenovirus into the common bile duct can induce safe and efficient transgene expression in hepatocytes without causing considerable adverse effects, supporting the feasibility of adenovirus-mediated gene transfer into hepatocytes in clinical settings by means of endoscopic retrograde cholangiography.