The P2X₁ receptor is a fast ATP-gated cation channel expressed in blood platelets, where its role has been difficult to assess due to its rapid desensitization and the lack of pharmacological tools. In this paper, we have used P2X₁^−/− and wild-type mouse platelets, treated with apyrase to prevent desensitization, to demonstrate the function of P2X₁ in the response to thrombogenic stimuli. In vitro, the collagen-induced aggregation and secretion of P2X₁-deficient platelets was decreased, as was adhesion and thrombus growth on a collagen-coated surface, particularly when the wall shear rate was elevated. In vivo, the functional role of P2X₁ could be demonstrated using two models of platelet-dependent thrombotic occlusion of small arteries, in which blood flow is characterized by a high shear rate. The mortality of P2X₁^−/− mice in a model of systemic thromboembolism was reduced and the size of mural thrombi formed after a laser-induced vessel wall injury was decreased as compared with normal mice, whereas the time for complete thrombus removal was shortened. Overall, the P2X₁ receptor appears to contribute to the formation of platelet thrombi, particularly in arteries in which shear forces are high.