There is strong evidence that antitumor activity of interleukin-12 (IL-12) in vivo is mediated, in part, through interferon (IFN gamma) produced by IL-12-stimulated natural killer and T cells. Since IFN gamma and tumor necrosis factor alpha (TNF alpha) have been reported to synergize in antitumor effects in a number of models, we decided to examine whether the combined treatment with recombinant mouse IL-12 and recombinant human TNF alpha would produce similar effects. The efficacy of the combined IL-12/TNF alpha immunotherapy was evaluated in three tumor models in mice: B16F10 melanoma, Lewis lung (LL/2) carcinoma and L1 sarcoma. Intratumoral daily injections of 1 microgram IL-12 in combination with 5 micrograms TNF alpha into B16F10-melanoma-bearing mice resulted in a significant retardation of the tumor growth as compared with that in controls and in mice treated with either cytokine alone. Similar effects were obtained using 0.1 microgram IL-12 and 5 micrograms TNF alpha in LL/2 carcinoma and L1 sarcoma models. Antitumor activity against L1 sarcoma was still preserved when TNF alpha at a low dose (1 microgram) was combined with 0.1 microgram IL-12 and applied for a prolonged time. Potentiation of antitumor effects, which was observed in IL-12/TNF alpha-based immunotherapy, could result from at least three different mechanisms, partly related to stimulation of IFN gamma and TNF alpha production in treated mice: (a) direct cytostatic/cytotoxic effects on tumor cells, (b) induction of antitumor activity of macrophages, and (c) inhibition of blood vessel formation in the tumor. Our studies demonstrate that combination tumor immunotherapy with IL-12 and TNF alpha may be more effective than single-cytokine treatment, and suggest possible mechanisms by which IL-12 and TNF alpha may exert potentiated therapeutic effects against locally growing tumors.