The role of macrophages in rat mixed leukocyte cultures was investigated. Previous studies indicated that macrophages in the responding cells suppressed the proliferative and cytotoxic responses. When, in the present study, macrophages were depleted from the stimulating cells, the proliferative and cytotoxic responses were greatly depressed. Reconstitution of the macrophage-depleted cultures with low concentrations of irradiated peritoneal exudate cells of the stimulator but not the responder haplotype resulted in the generation of strong cytotoxic responses. Further, highly purified stimulator peritoneal macrophage populations induced the generation of strongly cytotoxic effector cells, which suggested that macrophages are the predominant stimulating cells in rat mixed leukocyte cultures.