Progress to uncover the molecular and cellular regulators that govern human hematopoietic stem cell (HSC) fate has been impeded by an inability to obtain highly purified fractions of HSCs. We report that the rhodamine 123 (Rho 123) dye effluxing fraction of the Lin-CD34+CD38- population contains SCID-repopulating cells (SRCs) capable of long-term repopulation in primary nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Purification based on Rho uptake led to a 4-fold enrichment of SRCs in the Lin-CD34+CD38- fraction, with a frequency of 1 SRC in 30 Lin-CD34+CD38-Rholo cells. The Lin-CD34+CD38-Rholo fraction also possesses long-term self-renewal capacity as measured by serial transplantation totaling more than 20 weeks. We conclude that Rho dye efflux provides an additional means of purifying human HSCs in the quest to achieve homogeneous populations of primitive cells for both experimental and therapeutic applications.