Treatment with a combination of cytokines and chemotherapy can effectively stimulate the release of hematopoietic stem cells (HSC) into the peripheral blood (PB), which can then be harvested for transplantation. The cell cycle status of the harvested HSC from mobilized PB (MPB) is of interest because of the impact that cell cycling may have on optimizing the conditions for ex vivo expansion, retrovirus-mediated gene transfer, and the engraftment of transplanted tissues. Therefore, we characterized the cell cycling status of mobilized HSC from mice and humans. The murine HSC, which express the phenotype c-kit⁺ Thy-1.1^lo Lin^−/lo Sca-1⁺, were purified from PB, bone marrow (BM), and spleen after the mice were treated with the mobilizing regimen of granulocyte colony-stimulating factor (G-CSF ) or a combination of cyclophosphamide (CTX) and G-CSF. Human HSC (CD34⁺ Thy-1⁺ Lin⁻) and progenitor cells (CD34⁺ Thy-1⁻ Lin⁻) were isolated from the BM of untreated healthy volunteers and from MPB of healthy volunteers and patients treated with G-CSF or a combination of CTX and GM-CSF. Cell cycle status was determined by quantitating the amount of DNA in the purified cells after staining with the dye Hoechst 33342. Fluorescence-activated cell sorting analysis of the progenitor cells from the murine and human samples showed an unexpected finding, ie, virtually none of the cells from the MPB was cycling. The G0/G1 status of HSC from MPB was surprising, because a significant proportion of HSC from BM are actively proliferating and, after mobilization, the HSC in the spleen and BM were also actively cycling.