Bone marrow cells (BMC) flushed from femora of Lewis rats were cultured in Dulbecco's modification of Eagle's medium supplemented with mouse L929 cell supernatant as a source of colony-stimulating factor (CSF). Differentiation of macrophage progenitor cells into macrophages (M phi) and expression of various markers were kinetically assessed. The proportion of M phi increases from approximately 4% in freshly isolated BMC to 100% after 7-8 days of cell culture. These cells, termed bone marrow cell-derived macrophages (BMDM phi), adhere to and spread on plastic surface; exhibit M phi morphology; stain intensely for nonspecific esterase; are able to phagocytose latex particles, IgG-sensitized erythrocytes, and C3-coated red cells; and express receptors for IgG and C3. A subpopulation of BMDM phi expresses MHC class II antigens as demonstrated by immunofluorescence using MRC OX6 and MRC OX17 monoclonal antibodies which recognize antigens coded in the I-A or I-E subregion of the MHC, respectively. Collectively, our results show that supernatant from mouse L929 cells supports and is continuously required for proliferation and differentiation of rat BMC into typical M phi, and suggest that mouse CSF cross-reacts with the putative receptor on rat M phi.