Thromboxane A2, a potent vasoconstrictor and platelet agonist, is an evanescent cyclooxygenase product of arachidonic acid. Assessment of thromboxane biosynthesis commonly relies upon analysis of the stable but biologically inactive hydration product, thromboxane B2. However, measurement of this compound in plasma is readily confounded by platelet activation ex vivo. We have identified 11-dehydro-thromboxane B2, 11-dehydro-13,14-dihydro-15-keto-thromboxane B2, and 2,3-dinor-thromboxane B2 as enzymatic products of infused thromboxane B2 in the human circulation. Biosynthesis of deuterated standards permitted the development of quantitative analyses for these compounds, employing capillary gas chromatography-negative ion chemical ionization-mass spectrometry. We thus established that the postinfusion half-lives of 11-dehydro-thromboxane B2 and the keto-dihydro metabolite approximated 1 hour, while that of the dinor metabolite ranged from 15 to 17 min. Combined analysis of short- and long-lived enzymatic metabolites of thromboxane B2 promises to bypass the problem of ex vivo platelet activation and enhance the likelihood of relating a discreet clinical event to an alteration in the biosynthesis of thromboxane A2 in the human circulation.