The chemokine stromal derived factor-1α (SDF-1α) has been implicated recently in the chemotaxis of primitive human hematopoietic cells, suggesting that pluripotent human stem cells express the SDF-1α receptor, CXCR4. By using flow cytometry and confocal microscopy, we have identified and isolated primitive subsets of human CXCR4⁺ and CXCR4⁻ cells. Distinctions in the progenitor content and response to SDF-1α in vitro indicate that CXCR4⁺ and CXCR4⁻ cells represent discrete populations of primitive blood cells. The i.v. transplantation of these subfractions into immune-deficient mice established that both possess comparable engraftment capacity in vivo. Human myeloid, lymphoid, and primitive CD34⁺ CXCR4⁺ cells were present in chimeric animals transplanted with either subset, indicating that CXCR4⁺ and CXCR4⁻ stem cells have equivalent proliferative and differentiative abilities. Our study indicates that the human stem cell compartment is heterogeneous for CXCR4 expression, suggesting that the relationship between CXCR4 expression and stem cell repopulating function is not obligatory.