A monoclonal antibody, AN-18.17.24, specific for murine interferon-gamma (IFN-gamma) was produced by immunizing Wistar rats with IFN-gamma secreted by a T-cell lymphoma, L12-R4, upon stimulation with phorbol myristic acetate (PMA). Antiviral activity as well as tumoricidal activation induced by PMA-stimulated L12-R4 cell supernatant or by Con A-stimulated normal spleen cells were neutralized at the same extent by AN-18 monoclonal antibody. Moreover, depletion experiments showed that inhibition of tumoricidal macrophage activation must be ascribed to the direct binding of the IFN-gamma molecule by AN-18 MAb and not to the interference of the monoclonal antibody with the cell surface IFN-gamma receptor. These studies conclusively demonstrate that in supernatants of T lymphocytes stimulated with polyclonal activators IFN-gamma was the only molecule responsible for macrophage activation in tumor cell killing.