We have investigated the role of adenosine diphosphate (ADP) receptors in the adhesion, activation, and aggregation of platelets perfused over immobilized von Willebrand factor (VWF) under high shear stress. Blocking P2Y₁ prevented stable platelet adhesion and aggregation, indicative of a complete inhibition of α_IIbβ₃ activation, and decreased the duration of transient arrests from 5.9 seconds ± 2.8 seconds to 1.2 seconds ± 0.8 seconds; in contrast, blocking P2Y₁₂ inhibited only the formation of larger aggregates. Moreover, blocking P2Y₁ decreased the proportion of platelets showing early intracytoplasmic Ca⁺⁺ elevations (α/β peaks) from 20.6% ± 1.6% to 14.6% ± 1.5% (P < .01), and the corresponding peak ion concentration from 1543 nM ± 312 nM to 1037 nM ± 322 nM (P < .05); it also abolished the Ca⁺⁺ elevations seen in firmly attached platelets (γ peaks). Blocking P2Y₁₂ had no effect on these parameters, and did not enhance the effect of inhibiting P2Y₁. Inhibition of phospholipase C had similar consequences as the blocking of P2Y₁, whereas inhibition of Src family kinases abolished both type α/β and γ Ca⁺⁺ oscillations, although the former effect required a higher inhibitor concentration. Our results demonstrate that, under elevated shear stress conditions, ADP signaling through P2Y₁ may contribute to the initial stages of platelet adhesion and activation mediated by immobilized VWF, and through P2Y₁₂ to sustained thrombus formation.