In addition to possessing multilineage differentiation and self-renewal capabilities, pluripotent hematopoietic stem cells are believed to be mitotically quiescent and metabolically inactive. Fractions of human bone marrow (BM) CD34+ cells can be further enriched for primitive hematopoietic progenitor cells (HPC) by using a number of cell-surface markers. All of these fractions, however, contain cells that are still heterogeneous as far as their metabolic and mitotic activities are concerned. We therefore used Hoechst 33342 (Hst) to identify quiescent cells and Rhodamine 123 (Rh123) to identify metabolically inactive cells. CD34+ HstdimRh123dim (CD34+ d/d) and CD34+ HstbrightRh123bright (CD34+ b/b) cells were isolated by flow cytometry to examine the hematopoietic functions of mitotically and metabolically homogeneous progenitors. Cell-cycle status, progenitor cell content, maintenance of in vitro hematopoiesis, and long-term hematopoietic culture-initiating cell (LTHC-IC) content of CD34+ d/d and CD34+ b/b cells were compared with CD34+ HLA-DR-cells, a well-defined phenotype of primitive HPC. Whereas 99.2+/-0.5% of freshly isolated CD34+ d/d cells were in G0/G1 phase of the cell cycle, only 74.4+/-11.5% of CD34+ b/b and 75.6+/-1.1% of CD34+ HLA-DR-cells were in G0/G1. The number of multipotential progenitors (colony-forming units-granulocyte/erythroid/macrophage/megakaryocyte [CFU-GEMM]) detected in CD34+ d/d cells was twice that observed in CD34+ HLA-DR-cells and eight times that in CD34+ b/b cells. In stromal cell-free long-term cultures maintained for 10 weeks, production of assayable progenitors in cultures initiated with CD34+ d/d cells exceeded that detected in CD34+ HLA-DR-cultures by more than three-fold. Only in CD34+ d/d cultures were high proliferative potential colony-forming cell (HPP-CFC)-derived colonies detected over a period of 6 weeks. Limiting dilution analysis revealed that the frequency of LTHC-IC was highest among CD34+ d/d cells (7.2+/-3.3%), followed by a frequency of 4.5+/-4.8% for CD34+ HLA-DR-cells and 2.2+/-3.5% for CD34+ b/b cells. The primitive nature of HPC identified by CD34, Hst, and Rh123 was confirmed by the ability of as few as 200 murine marrow cells isolated by this technique to radioprotect and fully reconstitute lethally irradiated recipients. These results indicate that Hst and Rh123 staining can be used in combination with CD34 immunofluorescence to isolate a quiescent subpopulation of human primitive hematopoietic progenitor cells. Cells isolated by this technique appear to have functional properties associated with stem cells, suggesting that they may be ideal candidates for studies requiring primitive HPC, such as ex vivo expansion and somatic gene therapy.