Human colony-stimulating activity (CSA) may support the proliferation of both human and murine granulocyte-macrophage progenitor cells (CFU- C) or, in the case of human urinary CSA, may only stimulate murine bone marrow CFU-C. CSA produced in the culture media of monocytes and macrophages and phytohemagglutinin-stimulated lymphocytes from human peripheral blood was characterized for both human and mouse marrow CFU- C stimulating activities. During the initial phase of a long-term cultures of monocytes, both human- and mouse-active CSA (MnCM-HM) were produced. In later phases of culture, however, only mouse-active CSA (MnCM-M) was produced. Fractionation on Sephadex G-150 revealed two functionally distinct species from MnCM-HM and lymphocytes conditioned medium, a high molecular weight factor (MW greater than 150,000) which stimulated mouse but not human colony formation, and a low molecular weight species (MW 25,000–35,000) which was active against both mouse and human target cells. However, MnCM-M revealed only one high molecular weight species (greater than 150,000), active only on mouse marrow. The possible biologic significance of such an activity is discussed.