Gene therapy with IL-12 has been shown to elicit potent systemic antitumor response in a variety of tumors. Although direct intratumoral injection is the most commonly used delivery route for gene therapy of solid tumors, the skeletal muscle has been shown to be an ideal tissue for gene delivery to produce systemic gene expression. We have previously demonstrated that electroporation delivery of a reporter gene to muscle enhances the transfection efficiency and the level of gene expression by two to three logs. We report here that intramuscular (i.m.) injection of as little as 10 microg of the IL-12 DNA plasmid followed by electroporation prevents squamous cell carcinoma (SCCVII) tumor establishment in up to 40% of experimental animals and reduces the volume of established tumors by 75% compared to controls (P<.05). By comparison, there was no difference in tumor growth observed between IL-12 injection alone and injection of empty vector with or without electroporation. The induction of antitumor activity by i.m. electroporation delivery of the IL-12 gene is associated with an increase in IL-12 expression in muscle and serum. The level of IL-12 expression in muscle and serum was 1500 pg/tibialias muscle and 170 pg/mL serum, respectively, at day 6, after the gene was delivered by electroporation. In contrast, the level of IL-12 when the gene was injected without electroporation was hardly detectable after subtracting the background level of IL-12 detected in naïve mice. The high level of IL-12 expression led to a 170-fold induction of IFN-gamma expression in serum at day 6 after i.m. electroporation delivery of IL-12 DNA plasmid, which was equal to 1450 pg/mL in the serum. The induction of antitumor activity by i.m. electroporation delivery of the IL-12 gene also correlates with increased CD8+ T-cell population in peripheral blood but not in spleen. Our findings suggest that i.m. delivery of IL-12 gene using electroporation is an effective method of inducing a systemic antitumor response against SCC.