Efficient targeted gene delivery is essential for successful gene therapy. In this study, we examined the liver asanguineous perfusion method (LAP) for adenovirus-mediated gene transfer to the liver from the standpoints of efficiency, tissue-specificity and safety. The adenoviral vector containing the E. coli LacZ gene driven by the CAG promoter was delivered to the livers of rats by LAP. This method involves selective in situ perfusion, with the liver isolated by clamping of the afferent and efferent blood vessels to prevent adenoviral vector dissemination and genetic modification of nonhepatic organs. We demonstrated that gene transfer to the liver by LAP was not uniform, but more efficient than by intravenous (i.v.) or intraportal (i.p.) infusion, and caused no obvious liver damage or high mortality. As determined by specific histochemical staining and polymerase chain reaction, the amount of vector DNA transferred to the nonhepatic organs by LAP was significantly less than that transferred by the other two methods. Our data suggest that LAP is clearly superior to i.v. or i.p. infusion in terms of efficiency, specificity and safety of gene delivery to the liver. Further reduction in surgical risk is needed for the clinical application of gene therapy.