Macrophage-activating factors (MAF)3 were released by presensitized rat lymphocytes stimulated in vitro with the appropriate antigens. Different supernatants of presensitized rat lymphocytes specifically stimulated in vitro with several different mouse, dog, and rat tumor or normal cells were capable of rendering normal rat and mouse macrophages nonspecifically cytotoxic in vitro to their respective syngeneic tumor cells. The release of active mediators by rat lymphocytes sensitized in vivo was dependent upon immunologically specific recognition of an antigen in vitro. When rat lymphocytes were incubated in vitro with antigens unrelated to the in vivo sensitizing antigens, no release of MAF occurred. Once rat MAF was released, it activated both syngeneic (rat) and xenogeneic (mouse) macrophages to kill tumor cells in vitro. These activated marcophages destroyed all syngeneic tumor targets. Such cytotoxicity was obtained even when the cells used to elicit release of MAF were totally unrelated to the target tumor cells. The data thus demonstrated that MAF can cross strain and even species specificities and can activate macrophages to kill tumors in a nonspecific manner. The cytotoxicity mediated by in vitro activated mouse macrophages decreased with time once the macrophages were removed from MAF; and by 7 days postactivation, the macrophages were not cytotoxic. However, when incubated again with MAF, significant reactivation was observed. This suggested that activation of macrophages in vivo may be a continuous process of lymphocyte-macrophage interaction.