Oxidative metabolism of a 3-aroylthiophene, 1, by rat liver microsomes in the presence of mercaptoethanol as a trapping agent led to the isolation of four main compounds, 2-5, which have been isolated and characterized by UV, 1H NMR, and mass spectroscopy. They all derive from two primary metabolites, 2 and 3, which result from the nucleophilic addition of mercaptoethanol to a reactive, very electrophilic intermediate formed by sulfoxidation of the thiophene ring of 1. Further reactions of diastereoisomers 2 and 3 with mercaptoethanol led to compound 4 that is opened at the level of its thiophene ring and, eventually, to a final metabolite 5 resulting formally from the addition of mercaptoethanol on the 4, 5-double bond of the thiophene ring of 1. Compound 5 is very stable even in the presence of a large excess of mercaptoethanol. Similar reactions were observed upon microsomal oxidation of 1 in the presence of another thiol, N-acetylcysteine. Final metabolites 8a and 8b equivalent to 5 except for the replacement of its mercaptoethanol substituent with an N-acetylcysteinyl group were isolated and characterized by UV, 1H NMR, and mass spectroscopy. Interestingly, after treatment of rats with 1, metabolites 8a and 8b could be detected in urine, indicating that the successive reactions, that were observed in vitro after microsomal oxidation of 1 in the presence of a thiol-containing trapping agent, also occur in vivo, glutathione acting as a nucleophile in that case. These data provide clear evidence for the intermediate formation of a reactive. electrophilic thiophene sulfoxide in metabolic oxidation of 1 in vitro and in vivo. They also provide the first data on the complex reactivity of such thiophene sulfoxides, whose chemistry is poorly known, and on their fates in living organisms.