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 P2Y(1) prevented stable platelet adhesion and aggregation, indicative of a complete inhibition of alpha(IIb)beta(3) 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 P2Y(12) inhibited only the formation of larger aggregates. Moreover, blocking P2Y(1) decreased the proportion of platelets showing early intracytoplasmic Ca(++) elevations (alpha/beta 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 (gamma peaks). Blocking P2Y(12) had no effect on these parameters, and did not enhance the effect of inhibiting P2Y(1). Inhibition of phospholipase C had similar consequences as the blocking of P2Y(1), whereas inhibition of Src family kinases abolished both type alpha/beta and gamma Ca(++) oscillations, although the former effect required a higher inhibitor concentration. Our results demonstrate that, under elevated shear stress conditions, ADP signaling through P2Y(1) may contribute to the initial stages of platelet adhesion and activation mediated by immobilized VWF, and through P2Y(12) to sustained thrombus formation.