Human hematopoietic stem cells (HSCs) are commonly purified by the expression of cell surface markers such as CD34. Because cell phenotype can be altered by cell cycle progression or ex vivo culture, purification on the basis of conserved stem cell function may represent a more reliable way to isolate various stem cell populations. We have purified primitive HSCs from human umbilical cord blood (UCB) by lineage depletion (Lin^-) followed by selection of cells with high aldehyde dehydrogenase (ALDH) activity. ALDH^hiLin^- cells contained 22.6% ± 3.0% of the Lin^- population and highly coexpressed primitive HSC phenotypes (CD34⁺ CD38^- and CD34⁺CD133⁺). In vitro hematopoietic progenitor function was enriched in the ALDH^hiLin^- population, compared with ALDH^loLin^- cells. Multilineage human hematopoietic repopulation was observed exclusively after transplantation of ALDH^hiLin^- cells. Direct comparison of repopulation with use of the nonobese diabetic/severe combined immunodeficient (NOD/SCID) and NOD/SCID β2 microglobulin (β2M) null models demonstrated that 10-fold greater numbers of ALDH^hi-Lin^- cells were needed to engraft the NOD/SCID mouse as compared with the more permissive NOD/SCID β2M null mouse, suggesting that the ALDH^hiLin^- population contained committed progenitors as well as primitive repopulating cells. Cell fractionation according to lineage depletion and ALDH activity provides a viable and prospective purification of HSCs on the basis of cell function rather than cell surface phenotype. (Blood. 2004;104:1648-1655)