In order to examine the effect of recombinant growth factors on hemopoietic stem cells, these cells were enriched using wheat germ agglutinin (WGA) and monoclonal antibodies for lineage markers (Lin) such as B220, L3T4, Lyt-2, asialo GM1, Mac-1, and AL-21. Spleen colony-forming units (CFU-S) and in vitro colony-forming units were highly enriched in the fraction of WGA+Lin- spleen cells. To eliminate committed progenitor cells, spleen cells of 5-fluorouracil (5-FU)-treated mice were used. By this treatment, day-8 CFU-S disappeared but day-14 CFU-S were preserved. Day-14 CFU-S were also contained in the fraction of WGA+Lin- cells, which made up about 0.5% of total nucleated spleen cells. Moreover, this fraction contained primitive stem cells that could reconstitute the hemopoiesis of irradiated mice. Sorted WGA+Lin- spleen cells obtained from male 5-FU-treated mice were injected into lethally irradiated female mice. Southern hybridization using a mouse Y chromosome-specific probe showed that the bone marrow, spleen, and thymus of the recipients was reconstituted by male mouse-derived cells. When sorted WGA+Lin- spleen cells of the 5-FU-treated mice were cultured in vitro in the presence of recombinant interleukin 3 (IL-3), interleukin 6 (IL-6), and granulocyte colony-stimulating factor (G-CSF), colony formation was observed only in wells with IL-3, whereas unfractionated spleen cells formed colonies in the presence of IL-3, IL-6, or G-CSF. However, IL-6 but not G-CSF acted synergistically on enriched hemopoietic stem cells in the presence of IL-3. These data suggest that G-CSF or IL-6 did not affect primitive stem cells independently but showed the effect on these cells indirectly or synergistically with IL-3.