In the present study, to achieve more selective and efficient therapeutic gene expression in hepatoma cells, we compared the therapeutic efficacies of the retroviral vectors expressing the herpes simplex virus thymidine kinase (HSV-tk) gene by the a-fetoprotein (AFP) enhancer/promoter in the forward (LNAFE0.3TK) and reverse (LN[AFE0.3TK]R) orientation to the vector long terminal repeats. By Northern blotting, the level of the HSV-tk mRNA in LN[AFEO.3TK]R-infected HepG2 human hepatoma cells was much higher than that in LN[AFE0.3TK]R-infected cells. Consistent with this, LN[AFEO.3TK]R infection into HepG2 cells caused a greater cytotoxicity by ganciclovir exposure together with a stronger bystander effect than LNAFE0.3TK infection. In an animal model, intratumorous injection of LN[AFE0.3TK]R with ganciclovir treatment resulted in pronounced growth inhibition of HepG2 tumor. Thus, the reversely oriented therapeutic gene expression under the control of AFP enhancer/promoter is a possible candidate for the retrovirus-mediated gene therapy for hepatocellular carcinoma.