Hypercholesterolemia is one of the major contributing factors in atherosclerosis and the development of cardiovascular disease. Platelets from hypercholesterolemic rabbit have an increased cholesterol content and a hypersensitivity to endogenous aggregating agonists. Although rabbit has been widely used in studies of hypercholesterolemia, the precise role of platelet cholesterol in rabbit platelet activation has not been studied. In the present study, to determine the direct role of cholesterol on rabbit platelet activation, we examined the effect of in vitro modulation of cholesterol content on platelet activation. Cholesterol-depleted rabbit platelets by the treatment with methyl-beta-cyclodextrin showed decreased platelet aggregation by physiological agonists such as thrombin, adenosine diphosphate, and collagen. The inhibition of thrombin-induced aggregation in cholesterol-depleted platelets was restored by cholesterol repletion in platelets. The cholesterol depletion also inhibited Ca(2+) mobilization, which plays a pivotal role in the platelet activation induced by physiological agonists. We showed that the Ca(2+) influx pathway is strongly suppressed by cholesterol depletion more than Ca(2+) release from intracellular Ca(2+) stores in platelets stimulated with thrombin. Furthermore, platelet aggregation induced by PMA, a potent protein kinase C activator, was also depressed by cholesterol depletion. On the other hand, cholesterol enrichment in platelets augmented thrombin-induced aggregation and Ca(2+) mobilization. These findings suggest that cholesterol plays a critical role in regulating rabbit platelet activation, and provides fundamental information regarding hypercholesterolemia-mediated effects on cells in the rabbit model.