Antiviral and macrophage-priming activities in the supernatant medium of a subclone of a concanavalin A-stimulated mouse T-cell hybridoma were investigated. The two activities were associated with a molecular weight of approximately 50,000 and could not be separated by various approaches. Both activities were eliminated by a highly specific neutralizing antibody against mouse interferon-γ, but not by antibody against interferon-α and -β. The ratio of priming to antiviral activity in the hybridoma culture supernate was indistinguishable from the ratio obtained with mouse interferon-γ prepared by recombinant DNA technology. It was concluded from these data that the priming activity in hybridoma culture supernates was attributable to interferon-γ and that this mediator is one form of the lymphokine macrophage-activating factor. Interferon-γ was greater than 800 times more efficient at priming mouse macrophages for tumor cell killing than was a mixture of interferon-α and -β. This finding contributes to growing awareness that type II interferon may have greater immunoregulatory potential than type I interferons.