The Gi-coupled P2Y12 receptor plays a crucial role in platelet activation induced by ADP, and is also required for the action of a number of agonists whose receptors are couple to Gq, such as thromboxane A2. Stimulation of P2Y12 leads to the inhibition of adenylyl cyclase through the Gai subunit, and to the activation of phosphatidylinositol 3-kinase (PI-3K) and Rap1b through the bg subunit. Pharmacological inhibition of P2Y12 receptor results in the prevention of platelet aggregation induced by a number of agonists, including ADP and the the thromboxane A2 analogue U46619. In this study, we report that P2Y12 receptor is absolutely necessary for the stimulation of protein kinase C (PKC) and for PKC-directed protein phosphorylation. By using 32P-labeled human platelets, as well as by immunoblotting with anti-phospho(Ser) PKC substrates antibody, we analyzed the phosphorylation of pleckstrin, the main platelet substrate for PKC. As expected, we found that ADP-induced pleckstrin phosphorylation was totally suppressed by antagonists of the Gq-coupled P2Y1 receptor, but, surprisingly, we found that it was also completely prevented upon blockade of the P2Y12 receptor by AR-C69931MX or by 2-MeSAMP. Although we failed to directly detect any production of IP3 in ADP-stimulated platelets, we observed that ADP-induced Ca2+ release from intracellular stores was not affected by P2Y12 receptor antagonists. Moreover, AR-C69931MX or 2-MeSAMP strongly impaired pleckstrin phosphorylation induced by U46619, without affecting intracellular Ca2+ mobilization. These results indicated that blockade of the P2Y12 receptor did not interfere with phospholipase C stimulation, and that the reduced activation of PKC is probably not the consequence of a reduced production of diacylglycerol. Experiments performed with specific cell permeable inhibitors demonstrated that, in ADP-treated platelets, neither inhibition of adenylyl cyclase nor activation of PI-3K downstream P2Y12 receptor stimulation was responsible for PKC regulation. Subthreshold concentrations of PMA were able to restore ADP- or U46619-induced pleckstrin phosphorylation in the presence of P2Y12 antagonists, but were unable to restore platelet aggregation. Moreover, direct inhibition of PKC by Ro31-8220 prevented ADP-induced pleckstrin phosphorylation, but aggregation occurred normally. These results indicate that although PKC-directed protein phosphorylation is targeted by the P2Y12 receptor, it is not responsible for the essential contribution of this receptor to platelet aggregation. By contrast, we found that inhibition of platelet diacylglycerol kinase by R59949 completely reverted the inhibitory effect of AR-C69931MX or 2-MeSAMP on ADP- and U46619-induced activation of PKC, and completely restored platelet aggregation. These results suggest that diacylglycerol kinase may be regulated downstream the Gi-coupled P2Y12 receptor, and may be an important new regulator of platelet aggregation.