Interleukin 2 (IL-2) enhancement of herpes simplex virus-thymidine kinase (HSV-TK)/ganciclovir (GCV)-induced tumor killing was studied by cloning the human interleukin 2 gene into an HSV-TK-bearing adeno-associated viral (AAV) vector (TK/IL-2). The mouse hepatocellular carcinoma cell line Hepa 1-6 was used as a model in this study. We found that TK/IL-2-transduced Hepa 1-6 cells were more susceptible to ganciclovir treatment than tumor cells transduced with only TK in both nude mice and immunocompetent C57L/J mice. TK/IL-2-transduced tumors also showed shrinkage without GCV treatment. The tumor-killing effect of AAV-mediated TK/IL-2 gene transfer was further studied by inoculating animals with TK/IL-2- or TK-transduced tumor cells mixed with unmodified cells with or without GCV treatment. Although tumor growth in each group was inhibited, the best result was obtained from the TK/IL-2-transduced group without GCV treatment. In this group, 10% of the transduced tumor cells could eradicate the whole tumor in 50% of the animals tested as well as provide long-term protection against tumor cell rechallenge. When this group was treated with GCV, the antitumor effect of TK/IL-2 was reduced. We attribute this to the early ablation of transgene-bearing tumor cells by GCV treatment, which thus reduces the duration of IL-2 expression. We conclude that (i) TK/IL-2 plus GCV treatment generates a stronger tumor-killing effect than HSV-TK plus GCV and (ii) tumor killing of TK/IL-2 is more effective in non-GCV-treated animals than in GCV-treated animals.