Previous studies have suggested that Steel factor (SF) can influence the behavior of many types of hematopoietic progenitor cells both in vivo and in vitro, although whether these may include the most primitive populations of totipotent repopulating cells remains controversial. To approach this question, we measured the number of Sca1+Lin-WGA+ cells, the number of cells with demonstrable myeloid (long-term culture-initiating cell [LTC-IC]) or both myeloid and lymphoid (LTC-IC(ML)) potential in 4- to 5-week-old long-term cultures containing irradiated primary marrow feeder layers, and the number of multilineage long-term in vivo repopulating cells (competitive repopulating unit [CRU]) present in the marrow of W42/+ or W41/W41 mice compared to +/+ controls. There was no significant effect of either of these W mutations on the number of Sca1+Lin-WGA+ cells and, in W41/W41 mice, neither LTC-IC nor LTC-IC(ML) populations appeared to be affected. On the other hand, although W41/W41 and W42/+ cells could both be detected in the in vivo CRU assay, their numbers were markedly reduced (17- and seven-fold, respectively) in spite of the fact that both of these W mutant genotypes contained near normal numbers of day-9 and -12 colony-forming units-spleen (CFU-S). In vitro quantitation of erythroid (burst-forming units-erythroid [BFU-E]), granulopoietic (CFU-granulocyte/macrophage [CFU-GM]), multilineage (CFU-granulocyte/erythrocyte/monocyte/macrophage [CFU-GEMM]), and pre-B clonogenic progenitors (CFU-pre-B) also revealed no differences in the numbers (or proliferative potential) of any of these cells when W41/W41 or W42/+ and normal mice were compared, although day 3 BFU-E from both types of W mutant mice showed no response to the typical enhancing effect exerted by SF on their +/+ counterparts. Taken together, these findings are consistent with the view that SF activation of c-kit receptor-induced signaling events is not a rate-limiting mechanism controlling red blood cell production during normal development until hematopoietic cells differentiate beyond the day-3 BFU-E stage. Nevertheless, normal hematopoietic stem cells do appear to be responsive to SF, since their W mutant counterparts display a disadvantage in the in vivo setting which is exaggerated under conditions of hematopoietic regeneration. On the other hand, alternative mechanisms also appear to contribute to the regulation of hematopoietic stem cell numbers in vivo and to their detection as LTC-IC in vitro.