Interferon (IFN)-gamma was produced in cultures of human leukocytes by combined stimulation with 12-O-tetradecanoylphorbol 13-acetate (TPA) and phytohemagglutinin (PHA). IFN-gamma was purified by sequential adsorption and elution from controlled-pore glass and concanavalin A-Sepharose and by subsequent adsorptive removal of contaminating proteins on DEAE-Sephacel at pH 8.0. Treatment of such partially purified IFN-gamma preparations with the anionic detergent NaDodSO4 (0.1% at 20-25 degrees C) decreased biological activity to approximately 5-20%. When analyzed by NaDodSO4/polyacrylamide gel electrophoresis the bulk of IFN activity not destroyed by NaDodSO4 treatment was recovered from two peaks with apparent molecular weights of 20,000 and 25,000. The two activity peaks showed close correspondence with Coomassie blue-stained bands regularly demonstrable in purified supernatants from induced cultures but absent from culture supernatants from uninduced cells. Available evidence suggests that the two bands, isolated in pure form, represent subspecies of IFN-gamma. Native IFN-gamma was found to have a lower affinity for alkyl agarose columns than human IFN-alpha or IFN-beta did, suggesting that IFN-gamma is a relatively hydrophilic protein. Sulfhydryl-specific binding of native IFN-gamma to an Affi-Gel 501 column suggested that this IFN contains free sulfhydryl.