Delivery of transcription factors to cancer cells to reprogram gene expression may represent a novel strategy to augment the production of immune stimulatory cytokines and trigger a more potent antitumor response. In the present study, a bicistronic retroviral vector (AP2) was used to transduce B16-F0 melanoma cells with IFN regulatory factor (IRF)-3, which has been shown to activate type I IFN genes (IFN-beta and IFN-alpha) as well as other cytokines. Gene-modified B16 melanoma cells were inoculated s.c. into C57BL/6 syngeneic mice. In animals receiving IRF-3 B16 melanoma cells, tumors grew at a 4- to 5-fold reduced rate, and tumors that developed from these mice had a moderate-to-dense infiltration of inflammatory cells, whereas only low levels of lymphocyte infiltration were observed in mock-transduced B16 tumors. Furthermore, tumor growth was not inhibited in severe-combined immunodeficient mice after inoculation of IRF-3-expressing B16 cells, which suggested that IRF-3-mediated antitumor responses were dependent on a functional adaptive lymphocyte response. Interestingly, these in vivo effects on tumor growth correlated with higher mRNA expression of chemokines such as MIP-1beta, RANTES, and IP-10, as well as dramatic increases in vitro in the inducibility of cytokine mRNA such as IFN-beta, TNF-alpha and interleukin 6. Our results demonstrate that with weakly antigenic tumors such as B16 melanoma, IRF-3 gene transfer can mediate important antitumor responses. These findings suggest a novel role for IRF-3 as a potential molecular target for gene therapy of cancer.