Recently we showed that the P2Y1 receptor coupled to calcium mobilization is necessary to initiate ADP-induced human platelet aggregation. Since the thienopyridine compound clopidogrel specifically inhibits ADP-induced platelet aggregation, it was of interest to determine whether the P2Y1 receptor was the target of this drug. Therefore we studied the effects of clopidogrel and of the two specific P2Y1 antagonists A2P5P and A3P5P on ADP-induced platelet events in rats. Although clopidogrel treatment (50 mg/kg) greatly reduced platelet aggregation in response to ADP as compared to untreated platelets, some residual aggregation was still detectable. In contrast, A2P5P and A3P5P totally abolished ADP-induced shape change and aggregation in platelets from both control and clopidogrel-treated rats. A2P5P and A3P5P (100 μM) totally inhibited the [Ca2+]i rise induced by ADP (0.1 μM) in control and clopidogrel-treated platelets, whereas clopidogrel treatment had no effect. Conversely, the inhibition of adenylyl cyclase induced by ADP (5 μM) was completely blocked by clopidogrel but not modified by A2P5P or A3P5P (100 μM). A3P5P (1 m M) reduced the number of [33P]2MeSADP binding sites on control rat platelets from 907 ± 50 to 611 ± 25 per platelet. After clopidogrel treatment, binding of [33P]2MeSADP decreased to 505 ± 68 sites per platelet and further decreased to 55 ± 12 sites in the presence of A3P5P (1 m M). In summary, these results demonstrate that the platelet P2Y1 receptor responsible for the initiation of aggregation in response to ADP is not the target of clopidogrel. Platelets may express another, as yet unidentified, P2Y receptor, specifically coupled to the inhibition of adenylyl cyclase and necessary to induce full platelet aggregation, which could be the target of this drug.