Interleukin-3 (IL-3)-dependent mouse myeloid 32DC13 cells differentiate to neutrophils in response to granulocyte colony-stimulating factor (G-CSF). Introduction of the human c-fms gene, which encodes the receptor for CSF-1, into 32DC13 cells gave rise to variants that were able to proliferate in medium containing either murine IL-3 or human recombinant CSF-1, but were unable to differentiate to granulocytes in response to G-CSF. Unlike parental 32CD13 cells, CSF-1-responsive derivatives expressed nonspecific esterase when grown in CSF-1, but did not exhibit many other morphologic, immunologic, or functional properties of mononuclear phagocyte differentiation, or express murine CSF-1 receptors. Accelerated turnover of the human CSF-1 receptor was observed in response to CSF-1 and phorbol esters, but not after stimulation with IL-3 or bacterial lipopolysaccharide. Although both CSF-1 and IL-3 induced tyrosine phosphorylation of heterologous substrates in the dually responsive cells, differences in the patterns of substrate phosphorylation were observed in response to the two hematopoietins. We conclude that expression of the human CSF-1 receptor in 32DC13 cells not only induces CSF-1 responsiveness, but alters its phenotype in a way that prohibits granulocyte differentiation.