Using in vitro progenitor assays, serum-free in vitro cultures, and the nonobese diabetic/severe combined immune-deficient (NOD/SCID) ecotropic murine virus knockout xenotransplantation model to detect human SCID repopulating cells (SRCs) with multilineage reconstituting function, we have characterized and compared purified subpopulations harvested from the peripheral blood (PB) of patients receiving granulocyte colony-stimulating factor (G-CSF) alone or in combination with stem cell factor (SCF). Mobilized G-CSF plus SCF PB showed a 2-fold increase in total mononuclear cell content and a 5-fold increase in CD34-expressing cells depleted for lineage-marker expression (CD34+Lin−) as compared with patients treated with G-CSF alone. Functionally, G-CSF plus SCF–mobilized CD34+CD38−Lin−cells contained a 2-fold enhancement in progenitor frequency as compared with G-CSF–mobilized subsets. Despite enhanced cellularity and progenitor capacity, G-CSF plus SCF mobilization did not increase the frequency of SRCs as determined by limiting dilution analysis by means of unfractionated PB cells. Purification of SRCs from these sources demonstrated that as few as 1000 CD34+CD38−Lin− cells from G-CSF–mobilized PB contained SRC capacity while G-CSF plus SCF–mobilized CD34+CD38−Lin−cells failed to repopulate at doses up to 500 000 cells. In addition, primitive CD34−CD38−AC133+Lin−cells derived from G-CSF plus SCF–mobilized PB were capable of differentiation into CD34-expressing cells, while the identical subfractions from G-CSF PB were unable to produce CD34+cells in serum-free cultures. Our study defines qualitative and quantitative distinctions among subsets of primitive cells mobilized by means of G-CSF plus SCF versus G-CSF alone, and therefore has implications for the utility of purified repopulating cells from these sources.