To study the role of nitric oxide (NO) in regulating oxygen consumption by vessel walls, the oxygen consumption rate of arteriolar walls in rat cremaster muscle was measured in vivo during flow-induced vasodilation and after inhibiting NO synthesis. The oxygen consumption rate of arteriolar walls was calculated based on the intra- and peri-vascular PO₂ values measured by phosphorescence quenching laser microscopy. The peri-vascular PO₂ value of the arterioles during vasodilation was significantly higher than under control conditions, although the intravascular PO₂ values under both conditions were approximately the same. The inhibition of NO synthesis, on the other hand, caused a significant increase in arterial blood pressure and a significant decrease in arteriolar diameter. Inhibition of NO synthesis also caused a significant decrease in both the intra- and peri-vascular PO₂ values of the arterioles. The inhibition of NO synthesis increased the oxygen consumption rate of the vessel walls by 42%, whereas enhancement of flow-induced NO release decreased it by 34%. These results suggest that NO plays an important role not only as a regulator of peripheral vascular tone, but also as a modulator of tissue oxygen consumption by reducing oxygen consumption by vessel walls. In addition, enhancement of NO release during exercise may facilitate efficient oxygen supply to the surrounding high metabolic tissue.