New activated macrophage-specific antigen (AcM.1) detected by a monoclonal antibody has been described. The AcM.1 antigen was only detectable on activated macrophages induced by pyran and Corynebacterium parvum but not on resident or thioglycollate medium (TGC), proteose-peptone, and mineral oil-elicited macrophages, and not on blood monocytes and neutrophils. Activated macrophages induced by pyran and C. parvum, as expected, exhibited tumor cytotoxicity, whereas TGC-elicited macrophages did not show any cytotoxicity. Moreover, pretreatment of pyran-activated peritoneal exudate cells with AcM.1 antibody plus complement abolished approximately 80% cytotoxicity. Thus, this AcM.1 antigen detected by a monoclonal antibody is the first unique marker of activated tumoricidal macrophages and should provide a useful probe for investigating the mechanisms of activation or differentiation of macrophages.