Most patients with hepatocellular carcinoma have an elevated α-fetoprotein (AFP) level. This high level of AFP expression is transcriptionally controlled by the 5′-flanking sequence of the AFP gene. Using the 5′-flanking sequence as a promoter for the herpes simplex virus thymidine kinase (HSV-TK) gene in an adenoviral vector (Av1AFPTK1), the therapeutic efficacy of adenovirus-mediated HSV-TK gene transduction, followed by ganciclovir (GCV) administration, was studied in tumors in athymic nude mice. Av1AFPTK1 transduction of two cell lines demonstrated HSV-TK enzyme activity only in the AFP-producing cells (HuH7) and not in the AFP nonproducing cells (SK-Hep-1). As expected, only transduced HuH7 cells were killed by GCV treatment. Transduction by an adenoviral vector harboring a Rous sarcoma virus promoter and HSV-TK gene (Av1TK1) showed enzymatic activity and GCV killing in both cell lines. All HuH7 tumors that were transduced with either Av1AFPTK1 or Av1TK1 completely regressed after GCV treatment. On the other hand, there was complete regression of SK-Hep-1 tumors only when treated with Av1TK1 and GCV and not when treated with Av1AFPTK1 and GCV. Thus, cell-specific killing was achieved by adenoviral vector containing AFP promoter for the HSV-TK gene and GCV treatment.