The inhibitory effect of 6-(10-hydroxydecyl)-2,3-dimethoxy-5-methyl-1,4-benzoquinone (idebenone) on platelet aggregation was studied in rat and human platelets in vitro, and the mechanism of inhibition was examined in rat platelets. Idebenone inhibited the aggregation induced by collagen and thrombin in washed platelets, and by arachidonate and ADP in platelet-rich plasma (PRP). The inhibition was more prominent in collagen- and arachidonate-induced aggregation. In collagen-induced aggregation of human platelets, idebenone was 8-fold more potent than aspirin. In addition, idebenone inhibited prostaglandin synthesis and thromboxane B2 production, and also increased the cyclic AMP content in platelets. However, the concentration of idebenone required to inhibit thromboxane B2 production was much lower than that required to increase cyclic AMP. These results indicate that idebenone inhibits platelet aggregation by inhibiting thromboxane B2 synthesis rather than by increasing cyclic AMP content.