To investigate the crucial role of platelet-derived thromboxane A 2 (TXA 2) in initiating Ag-specific contact sensitivity (CS), a platelet-dependent CS model using genetically mast cell-deficient W/W v mice, was provided. In vivo treatment with BAYu3405, a TXA 2 receptor antagonist, markedly suppressed CS responses in a dose-dependent manner. This inhibitory effect occurred when BAYu3405 was administered before an early initiating phase, suggesting that TXA 2 may be a potent initiator of platelet-mediated CS responses. When platelets were pretreated with BAYu3405 in vitro, platelet aggregation as well as serotonin release, which is able to induce the early phase response allowing local recruitment of CS effector T cells due to direct activation of vascular endothelial cells, was inhibited. The addition of U46619, a TXA 2 agonist, or a mixture of platelets and thrombin-enhanced expression of both ICAM-1 and VCAM-1 on isolated mouse aortic endothelial cells, which was completely abolished by pretreatment with BAYu3405. Furthermore, intradermal injection of U46619 into the ear of platelet-depleted mice led to CS responses with marked expression of ICAM-1 and VCAM-1 on the vascular endothelium. These findings suggest that TXA 2 generated from platelets activated with Ag may mediate initiation of CS responses through inducing serotonin release from platelets and the subsequent aggregation and up-regulated expression of ICAM-1 and VCAM-1 on vascular endothelial cells.