Metabolic activation of the tetrahydro-thienopyridine antithrombotic prodrug, prasugrel, involves two steps: an esterase-dependent hydrolysis of its acetate function leading to thiolactone 6 and a cytochrome P450 (P450)-catalyzed oxidative cleavage of this thiolactone. This article shows that this second step involves the intermediate formation of a sulfenic acid 9 that has been trapped by dimedone during the metabolism of prasugrel by rat and human liver microsomes. The dimedone adduct has been characterized by mass spectrometry (MS) and (1)H and (13)C NMR spectroscopy. This article also describes the fate of the sulfenic acid intermediate in liver microsomes in the presence of various nucleophiles. Its reaction with a water-soluble phosphine cleanly leads to the corresponding thiol 7, which has been reported as the pharmacologically active metabolite of prasugrel. Its reaction with glutathione (GSH) leads to mixed disulfide 11, which may further react with GSH in excess to provide thiol 7. Experiments using microsomal incubations in the presence of (18)O(2) and (18)OH(2) have provided the first data on the mechanism of the P450-catalyzed oxidative cleavage of thiolactones such as 6. They indicate that sulfenic acid 9 is derived from a nucleophilic attack of H(2)O either directly on the electrophilic keto group of intermediate keto-sulfoxide 12, which is formed by P450-dependent S-oxidation of 6, or on the keto group of a cyclic sulfenic ester 13, which could derive from the rearrangement of 12. These data provide a first detailed mechanism for the metabolic activation of prasugrel to its pharmacologically active metabolites such as thiol 7.