We have found that the crude lymphokines, which prime the human monocyte-derived macrophage to generate H2O2 and exert microbicidal activity against intracellular Leishmania donovani, are rich in interferon (IFN)-gamma (600-3,000 U/ml). To determine the role of this specific lymphocyte product in macrophage activation, lymphokines were pretreated with a monoclonal antibody that neutralizes human IFN-gamma. Antibody exposure completely abolished the capacity of both mitogen- and antigen-stimulated lymphokines to either enhance macrophage H2O2 release or induce leishmanicidal activity. In addition, partially purified and pure recombinant human IFN-gamma were as effective as crude lymphokines in activating macrophages, and 3 d of treatment with 300 U/ml resulted in a seven- to eightfold increase in H2O2 generation and the intracellular killing of both L. donovani promastigotes and amastigotes. The ability of crude lymphokines to induce monocytes and macrophages from a patient with chronic granulomatous disease to kill L. donovani promastigotes was similarly abrogated by anti-IFN-gamma antibody, and could also be achieved by IFN-gamma alone. These results suggest that IFN-gamma is the key macrophage-activating molecule present within human lymphokines, and indicate that IFN-gamma can enhance both the oxygen-dependent and -independent antiprotozoal mechanisms of human mononuclear phagocytes.