The effects of a potent P2Y1 receptor antagonist, N6-methyl-2'-deoxyadenosine-3',5'-bisphosphate (MRS2179) on adenosine-5'-diphosphate (ADP)-induced platelet aggregation in vitro, ex vivo and on the bleeding time in vivo were determined. In suspensions of washed platelets, MRS2179 inhibited ADP-induced platelet shape change, aggregation and Ca2+ rise but had no effect on ADP-induced inhibition of adenylyl cyclase. Binding studies using the new radioligand [33P]MRS2179 showed that washed human platelets displayed 134+/-8 binding sites per platelet with an affinity (Kd) of 109+/-18 nM. Finally, intravenous injection of MRS2179 resulted in inhibition of rat platelet aggregation in response to ADP and prolonged the bleeding time, in rats or mice, as compared to controls. These results suggest this potent P2Y1 receptor antagonist to be a promising tool to evaluate the in vivo effects of pharmacologically targeting the P2Y1 receptor with a view to antithrombotic therapy.