We have developed a recombinant replication-defective adenovirus containing human alpha-fetoprotein (AFP) promoter/enhancer to direct cell type-specific expression of the herpes simplex virus thymidine kinase (HSVtk) gene to AFP-producing hepatocellular carcinoma (HCC) cells. After an in vitro infection by a recombinant adenovirus carrying the lacZ gene under the control of human AFP promoter/enhancer (AdAFPlacZ), an expression of the lacZ gene was demonstrated efficiently in AFP-producing HuH-7 and HepG2 cell lines, but not in AFP-nonproducing HLE and HLF cell lines, although lacZ gene expression was demonstrated in all these cell lines when infected with adenovirus vector carrying lacZ gene driven by the beta-actin-based promoter. Expression of the HSVtk gene by adenovirus, from AFP promoter/enhancer (AdAFPtk) induced the cells sensitive to ganciclovir (GCV) in the AFP-producing cell line efficiently, but not in AFP-nonproducing HLF hepatoma cells. An in vitro bystander effect was observed when only 10% of the cells were infected with AdAFPtk. These findings suggest that the AFP promoter/enhancer sequence can provide the tumor-specific activity for the therapeutic gene expression, and that the AdAFPtk vector induces the selective growth inhibition by GCV in the adenovirus-infected human hepatoma cells in vitro. Recombinant adenovirus transfer of the HSVtk gene under the control of tumor-specific promoter followed by GCV may have promise as a targeted in situ treatment for solid neoplasms.