Nitric oxide (NO) and prostaglandins (PG) together play a role in regulation blood flow during exercise. NO also regulates mitochondrial oxygen consumption through competitive binding to cytochrome c oxidase. Indomethacin both uncouples and inhibits the electron transport chain in a concentration-dependent manner, and thus inhibition of NO and PG may regulate both muscle oxygen delivery and utilization. The purpose of this study was to examine the independent and combined effects of NO and PG blockade (L-NMMA and indomethacin respectively) on mitochondrial respiration in human muscle following knee extension (KE) exercise. Mitochondrial respiration was measured ex-vivo by high resolution respirometry in saponin-permeabilized fibers following 6 min KE in control (CON, n=8), arterial infusion of LNMMA (n=4) and Indo (n=4) followed by combined inhibition of NO and PG (L-NMMA + Indo, n=8). ADP-stimulated state 3 respiration with substrates for complex I (glutamate, malate) was reduced 50% by Indo. State 3 O(2) flux with complex I and II substrates was reduced less with both Indo (20%) and L-NMMA + Indo (15%) compared to CON. The results indicate that indomethacin reduces state 3 mitochondrial respiration primarily at complex I of the respiratory chain while blockade of NO by addition of L-NMMA counteracts the inhibition of Indo. This metabolic effect in concert with a reduction of blood flow likely accounts for in-vivo changes in muscle O(2) consumption during combined blockade of NO and PG.