Summary. Alpha granule release plays an important role in propagating a hemostatic response upon platelet activation. We evaluated the ability of various agonists to cause α granule release in platelets. Alpha granule release was measured by determining P-selectin surface expression in aspirin-treated washed platelets. ADP-induced P-selectin expression was inhibited both by MRS 2179 (a P2Y1 selective antagonist) and AR-C69931MX (a P2Y12 selective antagonist), suggesting a role for both Gαq and Gαi pathways in ADP-mediated α granule release. Consistent with these observations, the combination of serotonin (a Gαq pathway stimulator) and epinephrine (a Gαz pathway stimulator) also caused α granule release. Furthermore, U46619-induced P-selectin expression was unaffected by MRS 2179 but was dramatically inhibited by AR-C69931, indicating a dominant role for P2Y12 in U46619-mediated α granule release. Additionally, the Gα12/13-stimulating peptide YFLLRNP potentiated α granule secretion in combination with either ADP or serotonin/epinephrine costimulation but was unable to induce secretion by itself. Finally, costimulation of the Gαi and Gα12/13 pathways resulted in a significant dose-dependent increase in α granule release. We conclude that ADP-induced α granule release in aspirin-treated platelets occurs through costimulation of Gαq and Gαi signaling pathways. The P2Y12 receptor plays an important role in thromboxane A2-mediated α granule release, and furthermore activation of Gα12/13 and Gαq signaling pathway can cause α granule release.