The participation of interferon-gamma in activation of murine macrophages for tumor cell lysis was investigated. Biochemically macrophage activation factor and interferon-gamma have not been separated. Antiviral titers correlated closely with macrophage activation in antigen- or mitogen-induced spleen cell supernatants. A monoclonal rat antibody that neutralized virus-induced interferon was also found to neutralize interferon-gamma in such supernatants. These monoclonal antibodies were coupled to CH-Sepharose 4B and used for absorption of antiviral activity from mitogen-induced spleen cell supernatants. Absorption of the interferon was paralleled by the reduction of the macrophage-activating capacity of the supernatants. Data from control absorptions supported the specificity of the absorption effect. These results indicate that interferon-gamma is required for activation of macrophages for tumor cell lysis. These results can be interpreted in two ways: (a) the monoclonal antibodies cross-react with interferon-gamma and with a mediator that is required for activation of macrophages for tumor cell lysis or (b) interferon-gamma itself is an essential cofactor for macrophage activation.