Macrophage-enriched peritoneal exudate cells from mice infected with Mycobacterium bovis BCG, macrophage-like tumor cells (PU 5-1.8), and peritoneal macrophages propagated in vitro with macrophage growth factor released tumoricidal activity into the culture medium within 2 to 3 h after stimulation with nanogram quantities of bacterial lipopolysaccharide. The cytotoxic activities from each of the macrophage culture supernatants eluted from diethylaminoethyl-Sephacel columns at a sodium chloride concentration of 200 mM exhibited a molecular weight of 50,000 to 60,000 as estimated by gel filtration, were stable at 56 degrees C for 30 min, and were active at a pH range of 6 to 10. A rabbit antiserum directed against serum-derived cytotoxic activity (tumor-necrotizing factor) from BCG-infected and lipopolysaccharide-challenged mice inhibited all of the cytotoxic activities generated in vitro. This suggests that the macrophage-derived cytotoxins are identical with serum-derived cytotoxic factor, which further implies that the macrophage is the cellular source of tumor-necrotizing factor.