Human monoblast-like histiocytic lymphoma cell line U937 was induced by a macrophage activating factor for O2- production (MAF-O) to produce O2- in response to phorbol myristate acetate stimulation. A MAF-O-producing human T-cell hybridoma, F4-29-4, was established which was also found to produce macrophage activating factor for glucose consumption (MAF-G) and colony stimulating factor (CSF) when assayed against mouse bone marrow cells. MAF-O could be successfully separated from CSF but not from MAF-G by phenyl-Sepharose CL-4B chromatography (Phenyl-EG-fraction). To differentiate MAF-O from MAF-G and to explore a route for large scale production of MAF-O and its structural elucidation, total messenger RNA was extracted from a human T-cell hybridoma, clone F4-29-4. This messenger RNA was fractionated on 5-30% sucrose gradient and each fraction was microinjected into Xenopus laevis oocytes. MAF-O activity was found in the supernatant of oocytes injected with messenger RNA sedimentated at about 13.0S, while MAF-G messenger RNA was found to be about 10.5S. The MAF-O activity, synthesized from the injected messenger RNA, was not neutralized with an excess amount of anti-human IFN-gamma anti-serum, suggesting that MAF-O is antigenically different from human IFN-gamma.