Macrophage-activating factor (MAF) was obtained from cultures of normal F344 rat lymphocytes incubated with insoluble concanavalin A. The MAF rendered macrophages from normal C57BL/6 mice cytotoxic against the syngeneic B16 melanoma and the allogeneic AC 15091. At the same time, normal syngeneic or allogeneic embryo cells were unharmed, even in the presence of susceptible tumor cells. Optimal MAF levels followed incubation of lymphocytes for 48 hr with Sepharose-bound concanavalin A. A 2-hr incubation of macrophages with MAF was sufficient to initiate activation, providing that 46 hr were allowed to elapse before tumor cells were added. The MAF activity was enhanced after heating the supernatant to 199 degrees. Control experiments largely excluded the possibility that residual unbound concanavalin A caused the observed macrophage-mediated tumoricidal effects.