The effects of recombinant rat stem cell factor (SCF/c-kit ligand) on murine megakaryocytopoiesis were studied using partially purified bone marrow cells derived from normal and 5-fluorouracil (5-FU)-treated mice in a serum-free culture system. SCF alone did not support the formation of megakaryocyte (M) and granulocyte-macrophage-megakaryocyte (GMM) colonies. However, the addition of SCF to cultures containing interleukin-3 (IL-3) resulted in a significant increase in the number of M and GMM colonies formed by bone marrow cells from normal mice, whereas IL-6 augmented only M colony growth. The stimulatory effect of SCF was approximately three to four times as high as that of IL-6 on the primitive progenitors capable of megakaryocytic-lineage expression derived from 5-FU-treated mice. In addition, SCF, but not IL-6, significantly increased the number of constituent cells in the individual M colonies supported by IL-3. On the other hand, SCF did not exert any effect on the size and DNA content of megakaryocytes in IL-3- dependent M and GMM colonies, whereas IL-6 enhanced the maturation of megakaryocytes. These results suggest that SCF stimulates the proliferative process in megakaryocytic progenitors and that the main activity of IL-6 is the promotion of megakaryocyte maturation.