The efficacy of combination treatment with actinomycin D (Act D), recombinant human tumor necrosis factor-alpha (TNF-alpha), and recombinant murine interferon-gamma (IFN-gamma) was examined on established MmB16 melanoma in mice. TNF-alpha alone had marginal effect in vitro on melanoma cells. However, when this cytokine was combined with either Act D or IFN-gamma, synergistic cytostatic/cytotoxic effects were observed. The highest cytotoxicity was demonstrated in cultures of melanoma cells in which all three agents together were added. In mice inoculated with 10(6) melanoma cells (into the footpad of the hind limb) and treated locally with Act D, TNF-alpha and IFN-gamma, beneficial therapeutic effects were found. When initiated 1 week after tumor cell inoculation, the 7-day treatment with all these agents administered together at daily doses: 0.2 microgram (Act D), 1 microgram (TNF-alpha), and 200 U (IFN-gamma) resulted in a significant delay of tumor progression in comparison to the therapy that included either Act D alone or TNF-alpha in combination with IFN-gamma. Side effects of such a treatment, both local and systemic, were negligible. The results of this study demonstrate that combination of regional chemotherapy (actinomycin D) and immunotherapy (TNF-alpha/IFN-gamma) may display higher efficacy than either treatment alone and may increase therapeutic index without augmenting toxic effects.