The antitumor effect and mechanism of action of IL-12 gene therapy combined with IFN-alpha gene therapy were investigated in tumor-bearing mice using renal and colon carcinoma models, Renca and CT26, respectively. Tumors were treated with murine IL-12 plasmid alone or in combination with IFN-alpha plasmid formulated with a polymeric interactive noncondensing (PINC) gene delivery system. Intratumoral injection of IL-12 DNA/polyvinyl pyrrolidone (PVP) alone induced rejection of 58 and 17% of Renca and CT26 tumors, respectively, whereas 25% (Renca) and 0% (CT26) rejection was observed in mice treated with IFN-alpha plasmid/PVP. Combination gene therapy of formulated plasmids, IL-12 with IFN-alpha, synergistically increased the antitumor response against Renca (100% tumor rejection) and CT26 (50%). In vivo depletion of leukocyte subsets indicated that CD8(+) T and NK cells were the primary effectors of the antitumor response induced by the combined cytokine gene therapy. Moreover, mice that rejected the primary tumors after combined treatment with IL-12 and IFN-alpha plasmid formulation developed protective immunity against a subsequent tumor challenge. Analysis of tumor-infiltrating leukocytes from mice treated with the combined IL-12 and IFN-alpha gene therapy showed upregulation of CD40 molecules on antigen-presenting cells (Mac-1(hi) cells). Finally, levels of mRNA for the chemokines IP-10 and TCA-3 were higher in tumors treated with the combination of cytokine plasmids than in tumors treated with either cytokine gene alone. These data provide evidence that IL12 gene therapy combined with IFN-alpha gene therapy synergistically induces regression of established tumors and may represent a novel therapeutic strategy for cancer treatment.