Activated human platelets synthesize prostaglandin (PG) E 2 , although at lower rate than thromboxane A 2 . PGE 2 acts through different receptors (EP1-4), but its role in human platelet function remains poorly characterized compared with thromboxane. We studied the effect of PGE 2 and its analogs on in vitro human platelet function and platelet and megakaryocyte EP expression. Platelets preincubated with PGE 2 or its analogs were stimulated with agonists and studied by optical aggregometry. Intraplatelet calcium mobilization was investigated by the stopped flow method; platelet vasodilator-stimulated phosphoprotein (VASP), P-selectin, and microaggregates were investigated by flow cytometry. PGE 2 at nanomolar concentrations dose-dependently increased the slope (velocity) of the secondary phase of ADP-induced platelet aggregation (EC 50 , 25.6 Ϯ 6 nM; E max of 100 Ϯ 19% increase versus vehicle-treated), without affecting final maximal aggregation. PGE 2 stabilized reversible aggregation induced by low ADP concentrations (EC 50 , 37.7 Ϯ 9 nM). The EP3 agonists, 11-deoxy-16,16-dimethyl PGE 2 (11d-16dm PGE 2 ) and sulprostone enhanced the secondary wave of ADP-induced aggregation, with EC 50 of 48.6 Ϯ 10 nM (E max , 252 Ϯ 51%) and 5 Ϯ 2 nM (E max , 300 Ϯ 35%), respectively. The EP2 agonist butaprost inhibited ADP-induced secondary phase slopes (IC 50 , 40 Ϯ 20 nM). EP4 stimulation had minor inhibitory effects. 11d-16dm PGE 2 alone raised intraplatelet Ca 2ϩ and enhanced ADP-induced Ca 2ϩ increase. 11d-16dm PGE 2 and 17-phenyltrinor PGE 2 (EP3 Ͼ EP1 agonist) at nanomolar concentrations counteracted PGE 1 -induced VASP phosphorylation and induced platelet microaggregates and P-selectin expression. EP1, EP2, EP3, and EP4 were expressed on human platelets and megakaryocytes. PGE 2 through different EPs finely modulates human platelet responsiveness. These findings should inform the rational selection of novel antithrombotic strategies based on EP modulation.