We have investigated whether adenovirally mediated gene transfer of the herpes simplex thymidine kinase gene to human hepatocellular carcinoma (HCC) cell lines can sensitize these cells to the prodrug ganciclovir and thereby provide a therapeutic option for this intractable cancer. Two replication-deficient adenoviruses encoding for the herpes simplex virus type-1 (HSV) thymidine kinase (TK) gene were generated in which expression of TK is under the control of either the human cytomegalovirus immediate early promoter (CMV) or the human alpha-fetoprotein (AFP) promoter/enhancer. We demonstrate that the combination of adenovirally mediated TK gene transfer and ganciclovir treatment effectively inhibits proliferation and causes cell death of HCC cells in vitro and that in vivo TK gene transfer and ganciclovir treatment inhibits hepatocellular tumor growth in a mouse model of this cancer. Furthermore, we show that expression of the TK gene can be restricted to those HCCs that express the tumor marker AFP through the incorporation of the AFP enhancer/promoter within an adenoviral vector.