A cloned variant of the EL-4 murine T-cell line treated with phorbol myristate acetate (PMA) releases a factor that activates macrophages for nonspecific tumor cytotoxicity. This macrophage activation factor (MAF) is both physicochemically (Mr 25,000; pH 2 stable) and biologically different from interferon-γ (IFN-γ). However, EL-4 MAF may represent a breakdown product or otherwise altered fragment of IFN-γ. We examined this possibility with a unique pair of hamster monoclonal antibodies against different epitopes of murine IFN-γ. Both antibodies inhibited IFN-γ-induced fibroblast antiviral activity; H21 but not H1 antibody also inhibited lymphokine (LK)-induced macrophage-mediated tumor cytotoxicity. Neither antibody, however, had any effect on the EL-4 MAF throughout a broad dose response. Moreover, passage through a H21 immunoaffinity chromatography column or addition of staphylococcal protein A and antibody completely inhibited LK-induced macrophage tumoricidal activity but did not affect the activity in EL-4 MAF. Identical effects in both fluid and solid phase were observed with polyclonal rabbit antisera to murine IFN-γ. Results with all of these antibodies strongly suggest that the EL-4 MAF and murine IFN-γ are antigenically distinct.