The importance of arachidonic acid metabolites (termed eicosanoids), particularly those derived from the COX-1 and COX-2 pathways (termed prostanoids), in platelet homeostasis has long been recognized. Thromboxane is a potent agonist, whereas prostacyclin is an inhibitor of platelet aggregation. In contrast, the effect of prostaglandin E₂ (PGE₂) on platelet aggregation varies significantly depending on its concentration. Low concentrations of PGE₂ enhance platelet aggregation, whereas high PGE₂ levels inhibit aggregation. The mechanism for this dual action of PGE₂ is not clear. This study shows that among the four PGE₂ receptors (EP1–EP4), activation of EP3 is sufficient to mediate the proaggregatory actions of low PGE₂ concentration. In contrast, the prostacyclin receptor (IP) mediates the inhibitory effect of higher PGE₂ concentrations. Furthermore, the relative activation of these two receptors, EP3 and IP, regulates the intracellular level of cAMP and in this way conditions the response of the platelet to aggregating agents. Consistent with these findings, loss of the EP3 receptor in a model of venous inflammation protects against formation of intravascular clots. Our results suggest that local production of PGE₂ during an inflammatory process can modulate ensuing platelet responses.