We describe a new lymphokine activity, macrophage cytotoxicity inducing factor 2 (MCIF2), in the T cell mitogen-induced supernatant of a murine T cell clone in long-term culture. MCIF2 has the following properties: it elutes from a Sephadex G-100 column in three m.w. forms (10, 34, and 100 KD); it is acid labile (pH 2 to 4) and heat sensitive (80 min at 56 degrees C); it is not constitutively secreted, coexists in the same supernatant with immune interferon (IFN-gamma), and synergizes with IFN-gamma for induction of tumoricidal and schistosomulicidal resident peritoneal mouse macrophages. We uncoupled this synergy and show that IFN-gamma serves as the first ("priming") and MCIF2 as the second ("triggering") signal for macrophage activation. Application of the lymphokines in the reverse order was ineffective. These data demonstrate a two-step mechanism of macrophage activation.