The development of novel cell-based therapies requires understanding of distinct human hematopoietic stem and progenitor cell populations. We recently isolated reconstituting hematopoietic stem cells (HSCs) by lineage depletion and purification based on high aldehyde dehydrogenase activity (ALDH hiLin- cells). Here, we further dissected the ALDH hi-Lin- population by selection for CD133, a surface molecule expressed on progenitors from hematopoietic, endothelial, and neural lineages. ALDHhiCD133+Lin- cells were primarily CD34+, but also included CD34-CD38-CD133 + cells, a phenotype previously associated with repopulating function. Both ALDHhiCD133-Lin- and ALDH hiCD133+Lin- cells demonstrated distinct clonogenic progenitor function in vitro, whereas only theALDH hiCD133+Lin- population seeded the murine bone marrow 48 hours after transplantation. Significant human cell repopulation was observed only in NOD/SCID and NOD/SCID β2M-null mice that received transplants of ALDHhiCD133+Lin- cells. Limiting dilution analysis demonstrated a 10-fold increase in the frequency of NOD/SCID repopulating cells compared with CD133+Lin- cells, suggesting that high ALDH activity further purified cells with repopulating function. Transplanted ALDHhiCD133+Lin- cells also maintained primitive hematopoietic phenotypes (CD34+CD38 -) and demonstrated enhanced repopulating function in recipients of serial, secondary transplants. Cell selection based on ALDH activity and CD133 expression provides a novel purification of HSCs with longterm repopulating function and may be considered an alternative to CD34 cell selection for stem cell therapies.