As part of our ongoing effort to identify a rich source of pluripotent progenitor cells for transplantation and gene therapy, we cultured single-sorted CD34+ subpopulations from different human hematopoietic tissues to assess the relationship between immunophenotype expression and functional characteristics. In combination with index sorting, single cell culture permits precise assessment of the colony efficiency (CE), growth characteristics, and replating potential (RP) of each individual phenotype without interference from other cell types. CD34+ cells from fetal liver (FL), fetal bone marrow (FBM), umbilical cord blood (UCB), adult BM (ABM), and mobilized peripheral blood (MPB) were sorted and cultured as single cells according to the coexpression of CD38. With the exception of FL, higher CEs were found among CD34+/CD38+ cells vs. CD34+/CD38- cells from the same cell source. However, colonies with dispersed growth pattern (DGP) and mixed growth pattern (MGP) were found predominantly in the CD34+/CD38- subsets. We next examined the functional characteristics of CD34+ subsets defined by three-color analysis and coexpression of CD38 and HLA-DR or CD38 and CDw90 (Thy-1). Using the combination CD34, CD38, and HLA-DR, the highest CEs, the highest percentages of colonies with DGP/MGP and the maximum RP were found in the CD34+/CD38-/HLA-DR+ subset among samples from FL, FBM, and UCB. The CEs and percentages of colonies with DGP/MGP of single-sorted CD34+/CD38-/HLA-DR+ cells were 72.7+/-11.8% (mean +/- standard deviation) and 20.1+/-10.4%, respectively, in FL; 60.9+/-11.1% and 11.5+/-5.4% in FBM; 57.0+/-16.5% and 24.1+/-7.3% in UCB; 27.2+/-12.8% and 9.0+/-4.9% in MPB, and 9.6+/-7.8% and 4.6+/-3.2% in ABM. Using the combination CD34, CD38, and CDw90(Thy-1), the subset with the highest CEs and highest percentages of colonies with DGP/MGP was found to be CD34+/CD38-/CDw90- in FL and FBM, but colonies with high RP were distributed evenly among CDw90+ and CDw90- subsets derived from FL, FBM, UCB, or MPB. We conclude that the CD34+/CD38-/HLA-DR+ subset contained the highest number of candidate stem cells among the various immunophenotypes, and that FL contained the highest concentration of CD34+ cells (11.4+/-7.5%) and the highest level of CD34+/CD38-/HLA-DR+ subsets (34.7+/-8.2%) among cells from various ontogenic age. Our estimate of candidate stem cells using single cell suspension culture correlated with that obtained by single cell long-term culture-initiating cells. CD34+/CD38-/HLA-DR+ cells from FL appear to represent the best targets for ex vivo stem cell expansion and genetic manipulation.