Despite increases in muscle sympathetic vasoconstrictor activity, skeletal muscle blood flow and O2 delivery increase during exercise in humans in proportion to the local metabolic demand, a phenomenon coupled to local reductions in the oxygenation state of haemoglobin and concomitant increases in circulating ATP. We tested the hypothesis that circulating ATP contributes to local blood flow and O2 delivery regulation by both inducing vasodilatation and blunting the augmented sympathetic vasoconstrictor activity. In eight healthy subjects, we first measured leg blood flow (LBF) and mean arterial pressure (MAP) during three hyperaemic conditions: (1) intrafemoral artery adenosine infusion (vasodilator control), (2) intrafemoral artery ATP infusion (vasodilator), and (3) mild knee-extensor exercise (∼20 W), and then compared the responses with the combined infusion of the vasoconstrictor drug tyramine, which evokes endogenous release of noradrenaline from sympathetic nerve endings. In all three hyperaemic conditions, LBF equally increased from ∼0.5 ± 0.1 l min−1 at rest to ∼3.6 ± 0.3 l min−1, with no change in MAP. Tyramine caused significant leg vasoconstriction during adenosine infusion (53 ± 5 and 56 ± 5% lower LBF and leg vascular conductance, respectively, P < 0.05), which was completely abolished by both ATP infusion and exercise. In six additional subjects resting in the sitting position, intrafemoral artery infusion of ATP increased LBF and leg vascular conductance 27 ± 3-fold, despite concomitant increases in venous noradrenaline and muscle sympathetic nerve activity of 2.5 ± 0.2- and 2.4 ± 0.1-fold, respectively. Maximal ATP-induced vasodilatation at rest accounted for 78% of the peak LBF during maximal bicycling exercise. Our findings in humans demonstrate that circulating ATP is capable of regulating local skeletal muscle blood flow and O2 delivery by causing substantial vasodilatation and negating the effects of increased sympathetic vasoconstrictor activity.