Plasma adenosine-5'-triphosphate (ATP) is thought to contribute to the local regulation of skeletal muscle blood flow. Intravascular ATP infusion can induce profound limb muscle vasodilatation, but the purinergic receptors and downstream signals involved in this response remain unclear. This study investigated: 1) the role of nitric oxide (NO), prostaglandins and adenosine as mediators of ATP induced limb vasodilation and 2) the expression and distribution of purinergic P2 receptors in human skeletal muscle. Systemic and leg hemodynamics were measured before and during 5-7 min of femoral intra-arterial infusion of ATP (0.45-2.45 µmol/min; mean±SEM) in 19 healthy, male subjects with and without co-infusion of NG-mono-methyl-L-arginine (L-NMMA; NO formation inhibitor; 12.3±0.3 mg/min), indomethacin (INDO; prostaglandin formation blocker; 613±12 µg/min) and/or theophylline (adenosine receptor blocker; 400±26 mg). During control conditions, ATP infusion increased leg blood flow (LBF) from baseline conditions by 1.82±0.14 L/min. When ATP was co-infused with either L-NMMA, INDO or L-NMMA+INDO combined, the increase in LBF was reduced by 14±6, 15±9, and 39±8 %, respectively (P<0.05) and was associated with a parallel lowering in leg vascular conductance and cardiac output and a compensatory increase in leg O₂ extraction. Infusion of theophylline did not alter the ATP induced leg hyperemia or systemic variables. Real time PCR analysis of the mRNA content from the vastus lateralus muscle of 8 subjects showed the highest expression of P2Y₂ receptors of the 10 investigated P2 receptor subtypes. Immunohistochemistry showed that P2Y₂ receptors were located in the endothelium of microvessels and smooth muscle cells, whereas P2X₁ receptors were located in the endothelium and the sacrolemma. Collectively, these results indicate that NO and prostaglandins, but not adenosine, play a role in ATP induced vasodilation in human skeletal muscle. The localization of the P2Y₂ and P2X₁ receptors suggest that these receptors may mediate ATP induced vasodilation in skeletal muscle.