We have recently shown that more than 90% of long-term culture initiating cells (LTC-IC) mobilized in the peripheral blood (PB) of normal individuals express HLA-DR and CD38 antigens and can sustain hematopoiesis for only 5 weeks. However, 10% of LTC-IC in mobilized PB are CD34⁺HLA-DR⁻ and CD34⁺CD38⁻ and can sustain hematopoiesis for at least 8 weeks. We now examine the ex vivo expansion potential of CD34⁺HLA-DR⁺ cells (rich in mature LTC-IC) and CD34⁺HLA-DR⁻ cells (rich in primitive LTC-IC) in granulocyte colony-stimulating factor (G-CSF ) mobilized PB progenitor cells (PBPC). Cells were cultured in contact with M2-10B4 cells (contact) or in transwells above M2-10B4 (noncontact) without and with interleukin-3 (IL-3) and macrophage inflammatory protein (MIP-1α) for 2 and 5 weeks. Progeny were evaluated for the presence of colony-forming cells (CFC) and LTC-IC. When CD34⁺HLA-DR⁺ PB cells were cultured in contact cultures without cytokines, a threefold expansion of CFC was seen at 2 weeks, but an 80% decrease in CFC was seen at week 5. Further, the recovery of LTC-IC at week 2 was only 17% and 1% at week 5. This confirms our previous observation that although CD34⁺HLA-DR⁺ mobilized PB cells can initiate long-term cultures, they are relatively mature and cannot sustain long-term hematopoiesis. In contrast, when CD34⁺HLA-DR⁻ mobilized PB cells were cultured in contact cultures without cytokines, CFC expansion persisted until week 5 and 49% and 11% of LTC-IC were recovered at week 2 and 5, respectively. As we have shown for steady state bone marrow (BM) progenitors, recovery of LTC-IC was threefold higher when CD34⁺HLA-DR⁻ PBPC were cultured in noncontact rather than contact cultures, and improved further when IL-3 and MIP-1α were added to noncontact cultures (96 ± 2% maintained at week 5). We conclude that although G-CSF mobilizes a large population of “mature” CD34⁺HLA-DR⁺ LTC-IC with a limited proliferative capacity, primitive CD34⁺HLA-DR⁻ LTC-IC present in mobilized PB have similar characteristics as LTC-IC from steady state BM: (1) they can be maintained in noncontact cultures containing IL-3 and MIP-1α for at least 5 weeks; (2) they are subject to the same proliferation inhibitory influences of contact with stroma. Since the absolute number of primitive LTC-IC (week 8 LTC-IC) per mL of G-CSF mobilized PB is similar to that per mL of steady state BM, these studies further confirm that G-CSF mobilized PBPC may have similar long-term repopulating abilities as steady state BM.