Using a serum-free culture system, we examined murine macrophage colony formation from bone marrow cells cultured in the presence of purified CSF-1, interleukin 3 (IL 3) or a combination of the two factors. CSF-1 supported macrophage and neutrophil-macrophage colony formation, whereas IL-3 supported the formation of various types of single lineage and multilineage colonies. CSF-1 supported more macrophage colonies from bone marrow cells of normal mice than IL 3, whereas in cultures of bone marrow cells of 5-fluorouracil-treated mice, IL 3 supported more macrophage colonies. A combination of CSF-1 and IL 3 resulted in granulocyte-macrophage (GM) colony formation that was equal to or greater than the sum of GM colony formation supported by the factors individually. The combination of CSF-1 and IL 3 resulted in significant increases in the size of both macrophage and neutrophil-macrophage colonies. Similar increases in colony size were observed when CSF-1 was added to cultures five days after incubation of marrow cells with IL 3. These data support the concept that some of the macrophage colony- forming cells that respond to IL 3 are more primitive than those that are sensitive to CSF-1.