Hemopoietin-1 (H-1) is known to act synergistically with CSF-1, a mononuclear phagocyte growth factor, to induce the development of primitive hemopoietic cells. To determine whether purified H-1 also acts on multipotent hemopoietic cells, its ability to act synergistically with interleukin-3 (IL-3) and erythropoietin (Epo) was tested in methyl cellulose cultures of murine bone marrow cells. In the presence of IL-3, H-1 increased the number of colonies formed by primitive, multipotent cells by approximately 30-fold. H-1 alone or H-1 plus Epo produced no colonies. Forty percent of the colonies induced by H-1 plus IL-3 contained cells that could be subcultured at least twice, whereas cells from colonies induced by IL-3 alone could not be similarly subcultured. Thus H-1 permits CSF-1 or IL-3 to act on cells more primitive than those acted on by either growth factor alone. The results indicate that H-1 acts on the most primitive hemopoietic cells yet shown to proliferate and differentiate in culture.