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One of the most important functions of the blood circulation is O₂ delivery to the tissue. This process occurs primarily in microvessels that also regulate blood flow and are the site of many metabolic processes that require O₂. We measured the intraluminal and perivascular pO₂ in rat mesenteric arterioles in vivo by using noninvasive phosphorescence quenching microscopy. From these measurements, we calculated the rate at which O₂ diffuses out of microvessels from the blood. The rate of O₂ efflux and the O₂ gradients found in the immediate vicinity of arterioles indicate the presence of a large O₂ sink at the interface between blood and tissue, a region that includes smooth muscle and endothelium. Mass balance analyses show that the loss of O₂ from the arterioles in this vascular bed primarily is caused by O₂ consumption in the microvascular wall. The high metabolic rate of the vessel wall relative to …