To test whether primitive hematopoietic stem cells (PHSCs) are stimulated by Steel (SI) factor (c-kit ligand) in vivo, donor mice were studied after three or seven daily injections of SI factor. PHSC activity was measured as long-term erythroid and lymphoid competitive repopulating ability. Cells to be tested (usually marrow or spleen cells from treated donors) were mixed with untreated competitor marrow that produces erythrocytes and lymphocytes that are genetically distinguishable from the donors by differences in hemoglobin (Hb) and glucosephosphate isomerase (GPI) markers. These cell mixtures were injected into lethally irradiated hosts, and after 111 to 293 days, functional abilities of donor PHSC populations were assessed and expressed as percentages of donor-type Hb and GPI in the host's circulating erythrocytes and lymphocytes, respectively. A striking increase in splenic PHSC activity occurred after seven daily injections of SI factor, with a much smaller increase after three daily injections. Both three and seven daily injections of SI factor slightly reduced marrow PHSC activity. Rapid cycling greatly increases PHSC vulnerability to 5-fluorouracil (5FU). To test whether SI factor stimulates PHSCs into rapid cycling, donor mice were given a dose of 5FU in addition to SI factor. The increase in splenic PHSCs after 7 days of treatment with SI factor occurred to a similar degree whether donors were or were not treated with 5FU on day 8. However, a dose of 5FU on day 4 of the SI factor treatments almost totally prevented the increase in splenic PHSC activity. Apparently this increased activity requires PHSC cycling throughout the period of SI factor treatment.