Culture supernatants from several human leukemic T cell lines were found to contain a macrophage activating factor which enhanced hydrogen peroxide release from human peripheral blood monocyte-derived macrophages. The macrophage activating factor from a T cell line, CCRF-CEM, was characterized biochemically and compared with interferon-gamma, which is also an immunological product of T cells and has a potent macrophage activating activity. In contrast to interferon-gamma, the macrophage activating factor in the culture supernatants bound to an anion exchanger and did not adsorb onto concanavalin A gel. Culture supernatants and active fractions from chromatographies were essentially devoid of anti-viral activity. Anti-human interferon-gamma monoclonal antibody also failed to neutralize the macrophage activating factor from CCRF-CEM. MAF was eluted in the fractions with molecular weight of 40,000 to 60,000 on gel filtration in the presence of a detergent and a salt. MAF was partially purified to about 1,300-fold by the methods described above: chromatography with anion exchangers and gel filtration. It was concluded that MAF from CCRF-CEM was biochemically and immunologically different from interferon-gamma.