Potassium release through ATP-sensitive potassium (K(ATP)) channels contributes to hypoxic vasodilation in the skeletal muscle vascular bed: It is uncertain whether K(ATP) channels on muscle cells contribute to the process. Potassium from muscle cells must cross the interstitial space to reach the vascular tissues, whereas that from vascular endothelium would have a higher concentration in venous blood than in interstitial fluid. We determined the effect of systemic hypoxia on arterial, venous, and interstitial potassium in the constant-flow-perfused gracilis muscles of anesthetized dogs. Hypoxia reduced arterial Po(2) from 138 to 25 and Pco(2) from 28 to 26 mmHg. Arterial pH and potassium were well correlated (r(2) = 0.9): Both increased in early hypoxia and decreased during the postcontrol. In denervated muscles, perfusion pressure decreased from 95 to 76 mmHg by the end of the hypoxic period; neither venous nor interstitial potassium was elevated. In innervated muscles, perfusion pressure increased from 110 to 172 mmHg by the 11th min of hypoxia and then decreased to 146 mmHg by the end of the hypoxic period; venous potassium increased from 5.0 to 5.3 mM, but interstitial potassium remained unchanged. Glibenclamide abolished both the increase in venous potassium and the hypoxic vasodilation in the innervated muscle. Thus skeletal muscle cells were unlikely to have contributed to the release of potassium, which was suggested to originate from vascular endothelium. The sympathetic nerve supply may play a direct or indirect role in the opening of K(ATP) channels under hypoxic conditions.