Macrophage cell-mediated cytolysis (M-CMC) of tumor cells may be measured by culturing target cells on macrophage monolayers and determining target cell survival by either Rb or H-thymidine incorporation. Using these techniques, one can demonstrate cytotoxicity of nonimmune human and rat macrophages to a variety of tumor cell lines. Toxicity becomes maximal 8 to 16 hr after attaching macrophages to plastic surfaces and disappears after 36 to 48 hr. Addition of Escherichia coli endotoxin at 48 hr restores macrophage toxicity. Culture of macrophages on plastic surfaces also results in the release, nonspecifically, of a soluble cytotoxin whose kinetics of release, disappearance, and reappearance after endotoxin all parallel the M-CMC. The amount of toxin released into cultures containing target cells is markedly reduced, suggesting that it is the mediator of the M-CMC and that it is utilized or absorbed during target destruction. Sephadex G-100 chromatography and treatment with heterologous anti-lymphotoxin can distinguish this toxin from lymphotoxin.