The anti-tumor effects of actinomycin D (Act D) and recombinant human tumor necrosis factor (TNF)-a have been studied on 4 established murine tumor cell lines: MmBI6 melanoma, Lewis lung (LL/2) carcinoma, L I sarcoma and L I 2 10 leukemia. During short-term incubation (24 hr) Act D produced dosedependent cytostatic/cytotoxic effects against MmB 16, LL/2 and L I tumor cells but did not reduce the viability of these cells even at high concentration (I 0 kg/ml), below a threshold of 3060%. However, L I 2 I0 leukemic cells were highly susceptible to Act D, and no viable cells were detected in cultures incubated with I pg/ml of Act D. TNF-cxalone, when used under the same culture conditions, had only a negligible effect on all cell lines tested. However, the combination of this cytokine with Act D produced synergistic cytotoxic effects against MmB 16, LL/2 and L I cells but not against LIZ10 leukemia cells. In an in vivo model of regional therapy in which tumor-bearing mice were treated with Act D and TNF-a, a correlation with in vitro results was observed. In mice bearing MmB I 6 melanoma, LL/2 carcinoma and L I sarcoma, the most potent anti-tumor effects were observed in mice treated with Act D and TNF-a together. This treatment led to a delay of tumor growth and induced complete tumor regression in some cases. On the contrary, TNF-a did not enhance the effect of Act D in mice injected with LIZ10 leukemia cells. Our results show that TNF-(U can potentiate the anti-tumor effects of Act D against tumors weakly susceptible to Act D and may be a useful adjuvant to chemotherapy in the local treatment of neoplasia.