Human hematopoietic stem cells (HSC) are commonly purified by the phenotypic expression of cell surface markers such as CD34. We have recently characterized a novel strategy to purify reconstituting HSC from human umbilical cord blood (UCB) by lineage depletion (Lin−) followed by selection of cells with high aldehyde dehydrogenase (ALDH) enzyme activity. Lin− cells with high ALDH activity (ALDHhiLin−) represented approximately 0.1% of total UCB mononuclear cells and demonstrated enriched expression of the primitive HSC markers CD34 (91.0±2.9%) and CD133 (70.9±4.0%). Most notably, clonogenic progenitor function and in vivo reconstituting ability in immune deficient mice were exclusive to the ALDHhiLin− population. Here, we have further purified the ALDHhiLin− population based on the expression of CD133, or prominen, a non-restricted surface molecule expressed on primitive progenitor cells of hematopoietic, endothelial, and neural epithelial lineages. ALDHhiCD133− and ALDHhiCD133+ cells, sorted to >95% purity, represented 14.7±2.1% and 23.2±4.3% of the total human UCB Lin− population respectively (n=6). Both ALDHhiCD133−Lin− and ALDHhiCD133+Lin− cells demonstrated clonogenic progenitor function in vitro. However, total colony production was significantly enhanced (p<0.05) in ALDHhiCD133−Lin− cells (1 CFU in 3.5 cells, n=5) when directly compared to ALDHhiCD133+Lin− cells (1 CFU in 10 cells, n=6). Human hematopoietic repopulation was consistently observed in the bone marrow, spleen, and peripheral blood of NOD/SCID (n=23) and NOD/SCID B2M null (n=27) mice transplanted with as few as 103 ALDHhiCD133+Lin− cells, whereas transplantation of up to 2x105 ALDHhiCD133−Lin− cells produced no detectable human engraftment. BM repopulation at limiting dilution demonstrated increased NOD/SCID repopulating ability elicited by ALDHhiCD133+Lin− cells when directly compared to CD133+Lin− cells not selected for ALDH activity. Repopulating ALDHhiCD133+Lin− cells differentiated into cells expressing markers for mature myeloid (CD33, CD14) and B-lymphoid (CD19, CD20) cells. ALDHhiCD133+Lin− cells also supported the maintenance of primitive cell phenotypes up to 8 weeks post-transplantation (2.4±0.7% CD34+CD38−, 5.5±0.6% CD34+CD133+, n=6) and the repopulating function of these cells are currently being confirmed by secondary transplantation. We are also investigating the ability of ALDHhiCD133+Lin− cells to mediate tissue repair in non-hematopoietic organs. Fractionation of human HSC based on combined expression of CD133 and high ALDH activity provides a rigorous selection of purified hematopoietic stem and progenitor cells that maintain primitive characteristics after transplantation, and may be considered a potential alternative to CD34+ cell isolation.