Homing and repopulation of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice by enriched human CD34+ stem cells from cord blood, bone marrow, or mobilized peripheral blood are dependent on stromal cellderived factor 1 (SDF-1)/CXCR4 interactions. Recently, human cord and fetal blood CD34+CD38-CXCR4- and CXCR4+ cells, sorted with neutralizing anti-CXCR4 monoclonal antibody (mAb), were shown to have similar NOD/SCID repopulation potential. Herein we report that human cord blood CD34+CXCR4+ (R4+) and CD34+CXCR4- (R4-) subsets, sorted with neutralizing anti-CXCR4 mAb, engrafted NOD/SCID mice with significantly lower levels of human cells compared with nonsorted and SDF-1-migrated CD34+ cells. Coinjection of purified cells with 10 μg anti-CXCR4 mAb significantly reduced engraftment of all CD34+ subsets, and 50 μg completely abrogated engraftment by R4- and CD34+ cells. Importantly, R4- cells harbor intracellular CXCR4, which can be rapidly induced to cell surface expression within a few hours. Moreover, 48 hours of cytokine stimulation resulted in up-regulation of both cell surface and intracellular CXCR4, restoring migration capacities toward a gradient of SDF-1 and high-level NOD/SCID repopulation potential. In addition, homing of sorted R4- cells into the murine bone marrow and spleen was significantly slower and reduced compared to CD34+ cells but yet CXCR4 dependent. In conclusion, R4- cells express intracellular CXCR4, which can be functionally expressed on the cell membrane to mediate SDF-1-dependent homing and repopulation. Our results suggest dynamic CXCR4 expression on CD34+ stem and progenitor cells, regulating their motility and repopulation capacities.