A recombinant adenovirus (AdVAFP1-IL2) containing the murine alpha-fetoprotein (AFP) promoter was constructed to direct hepatocellular carcinoma (HCC)-specific expression of the human interleukin-2 (IL-2) gene. In vitro testing of AdVAFP1-IL2 showed HCC-specific IL-2 gene expression three to four orders of magnitude higher in AFP-producing HCC lines compared to non-AFP producing non-HCC lines. The in vivo efficacy and tumor specificity of AdVAFP1-IL2 was evaluated compared to AdVCMV-IL2 (in which the IL-2 gene is driven by the strong constitutive cytomegalovirus promoter) in the treatment of established human HCC (Hep 3B/Hep G2) xenografts growing in CB-17/SCID mice. Intratumoral injection of AdV resulted in growth retardation and regression in a majority of established hepatomas, but with a much wider therapeutic index and less systemic toxicity using the AFP vector. This study illustrates the superiority of using transcriptionally targeted recombinant AdV vectors in cytokine-based gene therapy.