Although immunotherapy with bacillus Calmette Guérin (BCG) is an established adjuvant treatment for malignant melanoma, the mechanism of its role in this process is unclear. To investigate the possible contribution of tumor-inhibitory cytokines induced by BCG, B16F10 melanoma cell growth in culture was assessed in response to purified cytokines and conditioned media of BCG-stimulated splenocytes. Interferon-gamma (IFN gamma) was the most potent single agent (IC50 approximately 50 pg/ml). Tumor necrosis factor alpha was substantially weaker (IC50 > 10 ng/ml) but provided synergy with IFN gamma. None of the other cytokines such as interleukin-2 (IL-2), IL-4, IL-6, IL-10, IL-12, or granulocyte/macrophage-colony-stimulating factor had direct antitumor activity against B16F10 melanoma cells. However, when IL-2 and/or GM-CSF were combined with BCG either by exogenous addition or through endogenous production by novel cytokine-secreting recombinant BCG (rBCG), a substantial increase in INF gamma production by splenocytes was observed. Antitumor activity of this conditioned medium directly correlated with IFN gamma concentration and was completely blocked by neutralizing antibody to IFN gamma. These results suggest that BCG may exert part of its antitumor action on melanoma through the induction of IFN gamma, which can be greatly enhanced through the concomitant addition of IL-2 and/or GM-CSF. Furthermore, by utilizing rBCG that secrete these cytokines, it may be possible to potentiate the antitumor effect of BCG directly at the site of BCG inoculation.