Mouse peritoneal macrophages activated for tumor cytotoxicity by any of several in vivo or in vitro treatments released a soluble cytotoxin into culture fluids only after exposure to small amounts of bacterial lipopolysaccharides. This cytotoxic factor was physicochemically similar to the cytotoxic factor (tumor necrosis factor) in sera of BCG-infected mice injected with lipopolysaccharide. A rabbit antiserum against partially purified serum cytotoxic factor also inhibited the activity of macrophage-derived cytotoxic factor. Of special interest was the observation that rabbit anti-cytotoxic factor inhibited the cytotoxic activity of macrophages both in the presence and in the absence of exogenously added lipopolysaccharide. Inhibition was not complete but was consistent in all experiments. Thus, cytotoxic factor was implicated as a possible effector molecule in the nonspecific tumoricidal activity of activated macrophages.