We studied the capacity of bone marrow hemopoietic stem cells to form colonies (colony forming units, CFU) in the spleens of irradiated recipient mice following incubation of the cells with T cell-derived mediators in short-term culture in vitro. The mediators were a) stem cell-activating factor (SAF) present in crude form in the supernatant from phytohemagglutinin (PHA); stimulated lymphocytes, and b) immunoenhancing factor (IEF) obtained in partially purified form from the supernatant of antigen-stimulated lymphocytes and characterized by its ability to enhance antibody formation in vitro. Both SAF and IEF increased the number of CFU in the cultured bone marrow cell suspension. However, only SAF, and not IEF, significantly stimulated DNA synthesis in the cultured cells, Furhter more, SAF appears to activate CFU in vitro, rather than merely to promote the survival of active CFU stem cells. Experiments with SAF and bone marrow from different strains of mice indicated that the biologic activity was not restricted by K and Ia regions of H complex.