ADP plays a central role in regulating platelet function. It induces platelet aggregation via the activation of 2 major ADP receptors, P2Y₁ and P2Y₁₂. We have investigated the role of P2Y₁₂ in platelet adhesion and thrombus formation under physiological flow by using blood from a patient with a defect in the gene encoding P2Y₁₂. Anticoagulated blood from the patient and from healthy volunteers was perfused over collagen-coated coverslips. The patient’s thrombi were smaller and consisted of spread platelets overlying platelets that were not spread, whereas control thrombi were large and densely packed. Identical platelet surface coverage, aggregate size, and morphology were found when a P2Y₁₂ antagonist, N⁶-(2-methylthioethyl)-2-(3,3,3-trifluoropropylthio)-β,γ-dichloromethylene ATP (also known as AR-C69931 MX), was added to control blood. The addition of a P2Y₁ antagonist (adenosine-3′,5′-diphospate) to control blood resulted in small, but normally structured, thrombi. Thus, the ADP-P2Y₁₂ interaction is essential for normal thrombus buildup on collagen. The patient’s blood also showed reduced platelet adhesion on fibrinogen, which was not due to changes in morphology. Comparable results were found by using control blood with AR-C69931 MX and also with adenosine-3′,5′-diphospate. This suggested that P2Y₁₂ and P2Y₁ were both involved in platelet adhesion on immobilized fibrinogen, thereby revealing it as ADP dependent. This was confirmed by complete inhibition on the addition of creatine phosphate/creatine phosphokinase.