Immunological gene therapy of cancer relies heavily on the activation of T cells, but tumors with defects in MHC gene expression are not recognized by MHC-restricted T cells. To investigate the potential of cytokine genes for the therapy of MHC-negative tumors, we transduced B78H1, a class I-negative murine melanoma clone, with a polycistronic vector carrying murine interleukin (IL)-12 genes. The clones studied produced 400–25,000 pg/ml IL-12; their in vitro growth properties were similar to those of parental cells. A complete inhibition of growth was observed in vivo both after s.c. and i.v. administration of all IL-12 clones. IL-12-transduced cells were also used as a therapeutic vaccine in mice bearing micrometastases by nontransduced parental cells. A significant (80–90%) reduction in the number of lung nodules was obtained. Immunohistochemical analysis and studies in immunocompromised hosts showed that T cells and natural killer cells had a significant role in the elimination of IL-12-releasing cells. In situ hybridization with cytokine probes detected a strong increase in the proportion of leukocytes positive for IFN-γ, tumor necrosis factor α, IL-1β, and IFN-inducible protein 10 at the site of rejection of IL-12-engineered tumor cells. However, it was clear that the loss of in vivo growth was also due to T-cell- and natural killer cell-independent factors, possibly related to the antiangiogenic properties of IL-12. In conclusion, tumor therapy based on IL-12 gene transduction was effective on a MHC-negative metastatic tumor, suggesting a possible application to MHC-defective human neoplasms.