Optical spectroscopy was used to evaluate mitochondrial anoxia in the isolated perfused rat kidney by determining the percentage of cytochrome aa3 in the reduced state. Despite high levels of plasma flow (greater than 20 ml . g-1 . min-1) and venous PO2 (greater than 300 mmHg), 25--40% of cytochrome aa3 appeared to be in its reduced form. Agents that alter metabolism or transport primarily in renal cortex had little or no effect on the redox state of cytochrome aa3. These included 3-mercaptopicolinate, an inhibitor of gluconeogenesis; 2-tetradecylglycidic acid, an inhibitor of fat metabolism via carnitine-fatty acyltransferase, and acetazolamide. In contrast, a decrease in medullary transport induced by the loop diuretics bumetanide or furosemide produced an increase in cytochrome aa3 oxidation consistent with a decrease in transport-related oxygen consumption in the hypoxic area. The results suggest that substantial portions of the kidney, particularly in the renal medulla, may normally operate on the brink of anoxia despite high PO2 in artery and vein.