Metabolic cleavage of the CO-S bond of some thioesters RCOSR' with the formation of RCOOH requires a monooxygenase-dependent oxidative activation of this bond. The nature of the S-containing product(s) resulting from this cleavage remains unclear in most cases. This communication provides the first evidence for the formation of sulfenic acid intermediates 4a and 4b during the oxidative cleavage of the CO-S bond of thiolactone metabolites 2a and 2b of antithrombotic prodrugs, ticlopidine and clopidogrel, by rat and human liver microsomes. These intermediates have been trapped by dimedone, and the corresponding adducts 5a and 5b have been characterized by mass spectrometry (MS) and (1)H and (13)C NMR spectroscopy. Their formation is monooxygenase-dependent and almost completely inhibited by microsomal cytochrome P450 inhibitors. Moreover, they were also formed upon incubation with microsomes containing recombinant human P450 3A4, 3A5, 2C8, 2C9, 2C19, 2D6, or 1A2. In the presence of thiols such as mercaptoethanol, N-acetylcysteine, or glutathione, microsomal incubations of 2a led to mixed disulfides that have been characterized by MS and should result from reaction of 4a with these thiols. At high thiol concentrations, one observed in HPLC-MS the formation of a product exhibiting the MS expected for the previously described thiol metabolite 3a, a reduction product of 4a that has been reported as the pharmacologically active metabolite of ticlopidine. These data provide the first evidence for the formation of sulfenic acid reactive metabolites upon P450-catalyzed oxidative cleavage of thioesters. They also provide a first detailed mechanism for the previously described formation of pharmacologically active thiols such as 3a upon oxidative metabolism of ticlopidine and clopidogrel.