We have analyzed c-kit expression by hematopoietic progenitors from normal and 5-fluorouracil (5-FU)-treated mice by staining with monoclonal anti-c-kit antibody ACK-4. Marrow cells that were enriched for progenitors by a combination of metrizamide density separation and negative immunomagnetic selection with lineage-specific monoclonal antibodies (MoAbs) were separated into three populations based on the level of c-kit expression, c-kit(high), c-kit(low), and c-kit-. The majority of colony-forming cells from normal mice were in c-kit(high) population, whereas most of the progenitors from 5-FU-treated mice were in the c-kit(low) population. Optimal colony formation from c-kit(low) cells from 5-FU-treated mice required the interactions of at least two factors among interleukin-3 (IL-3), IL-11 and steel factor (SF) whereas colony formation from c-kit(high) cells of normal mice was supported well by IL-3 alone. Blast cells that were derived from 5-day culture of c-kit(low) post 5-FU cells were c-kit(high). These observations suggest that the primitive hematopoietic progenitors in cell cycle dormancy are c-kit(low) whereas actively cell cycling maturer progenitors are c- kit(high). Mature cells, with the exception of mast cells, derived from secondary culture of the c-kit(high) blast cells expressed little, if any, c-kit. These results are consistent with a model in which c-kit expression progresses from low levels on primitive, dormant multipotent progenitors to high levels on later, actively cycling progenitors, and finally, decreases to very low or undetectable levels on most mature blood cells, with the exception of mast cells.