The in vitro growth of tumor cells infected with mycoplasmas was suppressed by macrophages pretreated with interferon (IFN), but the growth of mycoplasma-free tumor cells was not suppressed. Pretreatment of macrophages with IFN plus mycoplasmas or their soluble factors either simultaneously or sequentially, IFN first and mycoplasmas second, but not in the reverse order, was effective in activating macrophages to suppress the growth of mycoplasma-free tumor cells. Macrophages from C3H/HeJ mice (which respond only slightly to lipopolysaccharide) were activated by IFN plus mycoplasmas or their soluble factor, and their action was not influenced by the addition of a lipopolysaccharide-neutralizing agent, polymyxin B. These results suggest that the macrophage-activating agent in mycoplasmas does not mimic lipopolysaccharide. The administration of mycoplasmas plus IFN to mice with ascitic or solid tumors resulted in the reduction of tumor growth. The survival rate of tumor-bearing mice was improved by the administration of mycoplasmas, and this was synergistically enhanced by the addition of IFN. These results indicate (a) that mycoplasmas can be useful as a biological response modifier, and (b) that care should be taken to prevent contamination with mycoplasmas in experiments on macrophage activation.