We performed the present study to define the in vitro hemopoietic activity of murine recombinant (r) granulocyte colony-stimulating factor (G-CSF) using murine hemopoietic culture systems of normal bone marrow cells, fetal liver cells, and spleen cells of 5-fluorouracil (FU)-treated mice. Recombinant G-CSF supported only neutrophil and/or macrophage colony formation by normal bone marrow cells. It did not enhance the formation of erythroid bursts in the fetal liver cell assay, but interleukin-3 (IL-3) did. Paradoxically, rG-CSF could support the colony formation of multilineage colonies as well as blast colonies from the spleen cells of 5-FU-treated mice, while r-granulocyte-macrophage colony-stimulating factor (GM-CSF) and r-erythropoietin (Ep) did not. When blast colonies, formed in the presence of G-CSF, were replated to dishes containing IL-3, they were able to differentiate along multilineage pathways. However, when they were replated to dishes containing rG-CSF, they could differentiate only into neutrophils and macrophages. Single cells transferred from blast colonies formed only neutrophil-macrophage colonies. These data indicate that rG-CSF had a direct effect on the growth and development of GM progenitors at a late stage and a significant effect on multipotential hemopoietic precursors. Although it remains to be clarified how G-CSF acts on multipotential stem cells, this unique effect is important in the understanding of its pluripotent hemopoietic activity in vivo.