Summary. ADP is important in propagating hemostasis upon its secretion from activated platelets in response to other agonists. Lipid rafts are microdomains within the plasma membrane that are rich in cholesterol and sphingolipids, and have been implicated in the stimulatory mechanisms of platelet agonists. We sought to determine the importance of lipid rafts in ADP-mediated platelet activation via the G protein-coupled P2Y1 and P2Y12 receptors using lipid raft disruption by cholesterol depletion with methyl-β-cyclodextrin. Stimulation of cholesterol-depleted platelets with ADP resulted in a reduction in the extent of aggregation but no difference in the extent of shape change or intracellular calcium release. Furthermore, repletion of cholesterol to previously depleted membranes restored ADP-mediated platelet aggregation. In addition, P2Y12-mediated inhibition of cAMP formation was significantly decreased upon cholesterol depletion from platelets. Stimulation of cholesterol-depleted platelets with agonists that depend upon Gαi activation for full activation displayed significant loss of aggregation and secretion, but showed restoration when simultaneously stimulated with the Gαz-coupled agonist epinephrine. Finally, Gαi preferentially localizes to lipid rafts as determined by sucrose density centrifugation. We conclude that Gαi signaling downstream of P2Y12 activation, but not Gαq or Gαz signaling downstream of P2Y1 or α2A activation, respectively, has a requirement for lipid rafts that is necessary for its function in ADP-mediated platelet activation.