ATP can be released from endothelial cells, and this release is increased by intraluminal flow in blood vessels. In the present study, the effect of extracellular ATP (1 µM) on flow-induced vasodilatation was investigated in isolated and pressurized rat small mesenteric arteries. In the absence of extracellular ATP, only 46% of arteries developed dilatation in response to flow, and this response was both transient and unstable. In marked contrast, with ATP present all vessels developed a prolonged and stable dilatation in response to flow. Even in the vessels that failed to respond to flow in the absence of ATP, dilatation could be stimulated once ATP was present. The ability of ATP to facilitate flow-induced vasodilatation was mimicked by UTP (1 µM), a P2Y agonist, or BzATP (10 µM), an agonist for P2X₁, P2X₇, P2Y₁₁. The involvement of P2X₇ was further supported by an inhibition effect of KN-62 (1 µM), a P2X₇ antagonist, on the action of BzATP. P2X₁ and P2X₃ were not involved, because their receptor agonist -methylene ATP had no effect. The facilitating effect of ATP on flow dilatation was also attenuated by the combined application of reactive blue 2 (100 µM), a P2Y antagonist, and suramin (100 µM), a nonselective P2X and P2Y antagonist. Furthermore, flow-induced dilatation obtained in the presence of ATP was reproducible. In contrast, in the additional presence of the ectonucleotidase inhibitor ARL 67156 (10 µM), although the first dilatation was normal, the responses to the second and later exposures to flow were greatly attenuated. Non-hydrolyzable ATP analogs ATP--s (1 µM) and AMP-PNP (10 µM) had similar effect to that of ARL 67156. These data suggest that ATP acts through both P2X and P2Y purinoceptors to facilitate flow-induced vasodilatation and that ectonucleotidases prevent this effect by degrading ATP on the endothelial cell surface.