Adenosine can induce vasodilation in skeletal muscle, but to what extent adenosine exerts its effect via formation of other vasodilators and whether there is redundancy between adenosine and other vasodilators remain unclear. We tested the hypothesis that adenosine, prostaglandins, and NO act in synergy to regulate skeletal muscle hyperemia by determining the following: (1) the effect of adenosine receptor blockade on skeletal muscle exercise hyperemia with and without simultaneous inhibition of prostaglandins (indomethacin; 0.8 to 1.8 mg/min) and NO (NG-mono-methyl-l-arginine; 29 to 52 mg/min); (2) whether adenosine-induced vasodilation is mediated via formation of prostaglandins and/or NO; and (3) the femoral arterial and venous plasma adenosine concentrations during leg exercise with the microdialysis technique in a total of 24 healthy, male subjects. Inhibition of adenosine receptors (theophylline; 399±9 mg, mean ± SEM) or combined inhibition of prostaglandins and NO formation inhibited the exercise-induced increase in leg blood flow by 14±1% and 29±2% (P<0.05), respectively, but combined inhibition of prostaglandins, NO, and adenosine receptors did not result in an additive reduction of leg blood flow (31±5%). Femoral arterial infusion of adenosine increased leg blood flow from ≈0.3 to ≈2.5 L/min. Inhibition of prostaglandins or NO, or prostaglandins and NO combined, inhibited the adenosine-induced increase in leg blood flow by 51±3%, 39±8%, and 66±8%, respectively (P<0.05). Arterial and venous plasma adenosine concentrations were similar at rest and during exercise. These results suggest that adenosine contributes to the regulation of skeletal muscle blood flow by stimulating prostaglandin and NO synthesis.