The preceding paper describes a new approach to the detection and assay of growth factors for developmentally early multipotent hemopoietic cells (Bartelmez et al., J. Cell. Physiol., 1985). This approach, involving measurement of the increase in the number of receptors for the mononuclear phagocyte specific hemopoietic growth factor (HGF), colony stimulating factor-1 (CSF-1), in cultures of developmentally early murine cells incubated with putative HGFs, has been used to define and assay hemopoietin-1. Hemopoietin-1 (Mr approximately 20,000) is found in the medium derived from serum-free cultures of cells of the human urinary bladder carcinoma line 5637. In contrast to both hemopoietin-2 and CSF-1, which also stimulate an increase in CSF-1 receptor numbers in cultures of developmentally early hemopoietic cells, hemopoietin-1 alone has no detectable effect. However, hemopoietin-1 exhibits dramatic synergism with CSF-1. In the presence of CSF-1, hemopoietin-1 stimulates the proliferation of developmentally earlier cells than those that respond to either CSF-1 alone or hemopoietin-2 alone or their combination. These cells proliferate for at least 3 days with no alteration of the average CSF-1 receptor density. However, by 5 days of incubation, the progeny of developmentally early hemopoietic cells that have proliferated in response to hemopoietin-1 + CSF-1 exhibit an approximately tenfold increase in the average CSF-1 receptor density per cell, which immediately precedes their differentiation to adherent mononuclear phagocytes. As hemopoietin-1 does not possess colony stimulating or burst promoting activities for murine bone marrow cells, but acts on multipotent hemopoietic cells, the analysis of the mechanism of its synergistic effects with HGFs such as CSF-1 are of special relevance to the regulation of early events in hemopoiesis.