In our previous studies using gene gun-mediated delivery of interleukin 12 (IL-12) cDNA in vivo, we observed T-cell-mediated regression of established murine tumors and demonstrated the induction of systemic immunity in test animals. In this study, we further characterized the antitumoral and anti-metastatic effect of this gene therapy approach by employing two murine metastatic mammary tumor models: the immunogenic TS/A adenocarcinoma and the weakly immunogenic 4T1 adenocarcinoma. In the TS/A model, gene transfer into the skin overlying an established intradermal tumor with an IL-12 cDNA expression vector resulted in complete tumor regression in 50% of mice followed by the development of immunological memory. In contrast, the growth of the intradermal 4T1 tumors was not affected by the IL-12 gene therapy protocol. However, this treatment resulted in a substantial reduction of spontaneous metastases in the lungs of 4T1 tumor-bearing mice and significantly prolonged their survival time. T cells were not required for this anti-metastatic effect, because it was also observed in nude mice and in mice depleted of CD4+ and CD8+ T cells. Tumor-draining lymph node cells obtained from 4T1 tumor-bearing mice treated with IL-12 cDNA exhibited increased natural killer (NK) activity and produced enhanced levels of interferon-gamma (IFN-gamma) compared with similar mice treated with luciferase cDNA. In addition, in vivo depletion of NK cells or neutralization of IFN-gamma resulted in partial suppression of the anti-metastatic effect of IL-12 gene therapy, suggesting the involvement of both NK cells and IFN-gamma in this effect.