We report here that a cancer gene therapy protocol using a combination of IL-12, pro-IL-18, and IL-1beta converting enzyme (ICE) cDNA expression vectors simultaneously delivered via gene gun can significantly augment antitumor effects, evidently by generating increased levels of bioactive IL-18 and consequently IFN-gamma. First, we compared the levels of IFN-gamma secreted by mouse splenocytes stimulated with tumor cells transfected with various test genes, including IL-12 alone; pro-IL-18 alone; pro-IL-18 and ICE; IL-12 and pro-IL-18; and IL-12, pro-IL-18, and ICE. Among these treatments, the combination of IL-12, pro-IL-18, and ICE cDNA resulted in the highest level of IFN-gamma production from splenocytes in vitro, and similar results were obtained when these same treatments were delivered to the skin of a mouse by gene gun and IFN-gamma levels were measured at the skin transfection site in vivo. Furthermore, the triple gene combinatorial gene therapy protocol was the most effective among all tested groups at suppressing the growth of TS/A (murine mammary adenocarcinoma) tumors previously implanted intradermally at the skin site receiving DNA transfer by gene gun on days 6, 8, 10, and 12 after tumor implantation. Fifty percent of mice treated with the combined three-gene protocol underwent complete tumor regression. In vivo depletion experiments showed that this antitumor effect was CD8(+) T cell-mediated and partially IFN-gamma-dependent. These results suggest that a combinatorial gene therapy protocol using a mixture of IL-12, pro-IL-18, and ICE cDNAs can confer potent antitumor activities against established TS/A tumors via cytotoxic CD8(+) T cells and IFN-gamma-dependent pathways.