The recent demonstration that platelets express a functional toll-like receptor 4 (TLR4) prompted us to explore the influence of TLR4 polymorphisms (Asp299Gly alone or in combination with Thr399Ile) on thromboxane A(2) (TXA(2)) biosynthesis in vivo. In 17 subjects with TLR4 polymorphisms versus 17 wild type (untreated with aspirin, matched for age, sex, and cardiovascular risk factors), intima-media thickness in the common carotid arteries was significantly lower. Average urinary excretion of 11-dehydro-TXB(2), an index of systemic biosynthesis of TX, was significantly reduced by 65%. The urinary excretion of 2,3-dinor-6-keto-prostaglandin F(1alpha), an index of systemic biosynthesis of prostacyclin, was marginally depressed but the prostacyclin/TXA(2) biosynthesis ratio was significantly higher than in wild type. Selective inhibition of cyclooxygenase 2-dependent prostacyclin (by rofecoxib or etoricoxib) was associated with increased urinary excretion of 11-dehydro-TXB(2) in carriers of TLR4 polymorphisms, but not in wild-type, suggesting a restrainable effect of prostacyclin on platelet function in vivo in this setting. Reduced TXA(2) biosynthesis may contribute to the protective cardiovascular phenotype of TLR4 polymorphisms.