The CXCR4/SDF-1 axis has been studied extensively because of its role in development and hematopoiesis. In acute myeloid leukemia (AML), elevated expression of CXCR4 has been shown to correlate with shortened survival. Hy-poxia increases CXCR4 in several tumor models, but the impact of reduced O(2) partial pressure (pO(2)) on expression and biologic function of CXCR4 in AML is unknown. We determined pO(2) in bone marrows of AML patients as 6.1% (+/-1.7%). At this pO(2), CXCR4 surface and total expression were up-regulated within 10 hours in leukemic cell lines and patient samples as shown by Western blotting, fluorescence-activated cell sorting, and microscopy. Interestingly, hypoxic cells failed to internalize CXCR4 in response to SDF-1, and upon reoxygenation at 21% O(2), surface and total expression of CXCR4 rapidly decreased independent of adenosine triphosphate or proteasome activity. Instead, increased pO(2) led to alteration of lipid rafts by cholesterol depletion and structural changes and was associated with increased shedding of CXCR4-positive microparticles, suggesting a novel mechanism of CXCR4 regulation. Given the importance of CXCR4 in cell signaling, survival, and adhesion in leukemia, the results suggest that pO(2) be considered a critical variable in conducting and interpreting studies of CXCR4 expression and regulation in leukemias.