Platelet activation is associated with platelet shape change, fibrinogen receptor activation, platelet aggregation, secretion of granule contents and phospholipase A2 (PLA2) activation. Agonist-induced PLA2 activation is important for liberation of arachidonic acid from membrane phospholipids, which is then subsequently converted to thromboxane A2 (TXA2) via the sequential effects of cyclo-oxygenase and thromboxane synthase respectively. Thus the generated TXA2 plays a very predominant role in potentiating the effects of other physiological platelet agonists like collagen, thrombin and ADP. ADP-induced TXA2 generation requires co-stimulation of the P2Y1 and P2Y12 receptors, fibrinogen receptor activation and subsequent outside-in signaling. We recently demonstrated that G-protein gated inwardly rectifying potassium channels (GIRKs) are important for P2Y12 receptor-mediated platelet functional responses, namely platelet aggregation, dense granule secretion and Akt phosphorylation. In this study, we evaluated the role of GIRK channels in ADP- mediated TXA2 production in human platelets. ADP-mediated TXA2 generation was inhibited in the presence of SCH23390 and U50488H in a concentration-dependent manner, with maximal inhibition occurring at 10 μM. At these concentrations of GIRK blockers, none of the other P2Y12 receptor-dependent platelet functional responses including aggregation, Akt phosphorylations were affected. We confirmed our findings by performing similar experiments with 2-MeSADP, a potent and selective P2Y1 and P2Y12 agonist. We observed that GIRK channel blockers inhibit 2-MeSADP-mediated TXA2 generation. These results suggest the existence of two different populations of GIRK channels;-one that plays an important role in TXA2 formation (sensitive to low concentrations of the GIRK blockers) and the other population that plays an important role in platelet aggregation (sensitive to higher concentrations of GIRK blocker). Experiments done in the presence of SKF38393, a structurally similar but inactive analog of SCH23390 did not have any effect on ADP or 2-MeSADP-induced TXA2 production in the non-aspirinated platelets. Finally, we also observed that arachidonic acid-induced TXA2 production is not affected by 10 μM concentrations of SCH23390 and U50488H. These data suggest that GIRK channel blockers inhibit TXA2 formation by interfering with agonist-induced PLA2 activation. Based on these findings we conclude that there exist different populations of GIRK channels, one that contributes to ADP- and 2-MeSADP-induced TXA2 and the other that contributes to P2Y12 receptor-dependent platelet functional responses. Also, each of the two populations of GIRK channels has varying susceptibilities to the GIRK channel blockers.