IL-12 is an immuno-regulatory cytokine that has been shown to generate a potent NK and Th1 response in a variety of laboratory models. However, the detailed immune development in the hepatic tumor model by IL-12-mediated gene therapy has not been clarified. In our previous study, intra-tumoral transfer of Adv.mIL-12 (5 x 10(8) pfu) to the MCA26 colon carcinoma liver tumor induced an effective anti-tumor response, extending the median survival time from 29 to over 54 days, while 25% of the animals became tumor-free after a single treatment. In this work, we show that NK cells are responsible for the early, and both NK and T cells for the long-term, Adv.mIL-12-induced immune response. Immunohistopathological analysis of the tumor and in vitro cytotoxicity study of the mononuclear cells of the liver show that NK cells are the first to infiltrate and mediate tumor cell killing, as early as 48 hr after Adv.mIL-12 treatment. In vivo and in vitro depletion of these cells completely abolishes this early anti-tumor response. This activity can be observed in both populations of conventional NK and NKT cells in vitro and in athymic nude mice in vivo. However, the early NK response alone is not sufficient. In vivo T-cell depletion in both the primary tumor treatment and the long-term survival rechallenge study reveals that T cells in addition to NK cells are required in the development of the long-term survival and immunity attributed to Adv.mIL-12 gene therapy in this orthotopic tumor model of colon carcinoma.