ATP has been proposed to play multiple roles in local skeletal muscle blood flow regulation by inducing vasodilation and modulating sympathetic vasoconstrictor activity, but the mechanism remain unclear. Here we evaluated the effects of arterial ATP infusion and exercise on limb muscle interstitial ATP and NE concentrations to gain insight into the interstitial and intravascular mechanisms by which ATP causes muscle vasodilation and sympatholysis. Leg hemodynamics and muscle interstitial nucleotide and norepinephrine (NE) concentrations were measured during: 1) femoral arterial ATP infusion (0.42±0.04 and 2.26±0.52 μmol/min; mean±SEM) and 2) one-leg knee-extensor exercise (18±0 and 37±2W) in 10 healthy, male subjects. Arterial ATP infusion and exercise increased leg blood flow (LBF) in the experimental leg from ~0.3 L/min at baseline to 4.2±0.3 and 4.6±0.5 L/min, respectively, whereas it was reduced or unchanged in the control leg. During arterial ATP infusion, muscle interstitial ATP, ADP, AMP and adenosine concentrations remained unchanged in both legs, but muscle interstitial NE increased from ~5.9 nmol/L at baseline to 8.3±1.2 and 8.7±0.7 nmol/L in the experimental and control leg, respectively (P<0.05), in parallel to a reduction in arterial pressure (P<0.05). During exercise, however, interstitial ATP, ADP, AMP and adenosine concentrations increased in the contracting muscle (P<0.05), but not in inactive muscle, whereas interstitial NE concentrations increased similarly in both active and inactive muscles. These results suggest that the vasodilatory and sympatholytic effects of intraluminal ATP are mainly mediated via endothelial prinergic receptors. Intraluminal ATP and muscle contractions appear to modulate sympathetic nerve activity by inhibiting the effect of NE rather than blunting its local concentration.