Direct evidence for the involvement of thiophene S-oxide as a key primary reactive intermediate in the metabolism of thiophene (1) in rats was obtained from the isolation of two diastereoisomeric thiophene S-oxide dimers, 4a and 4b, both in vitro (oxidation of thiophene with rat liver microsomes) and in vivo (isolation of 4a from rat urine). The structure of these dimers was established after an original preparation of identical samples by oxidation of thiophene with H2O2 and CF3COOH. In fact, the H2O2/CF3COOH system appeared to be the best oxidizing agent for the selective transformation of thiophene to its S-oxide. The complete determination of the structures of 4a and 4b was carried out for the first time by X-ray diffraction for the former and by a sequence of chemical reactions for the latter. The reported results indicate two fates for thiophene S-oxide in vivo : (i) its dimerization via a Diels-Alder reaction and (ii) its reaction with nucleophiles such as glutathione leading eventually to mercapturates. These results together with recent literature data on thiophene derivatives suggest that thiophene S-oxides, a class of reactive intermediates whose chemistry is still not well-known, could play a central role in the metabolism and toxic effects of thiophenes in mammals. This situation would be different from that observed in the metabolism of other aromatic compounds, such as benzene or furan, in which arene oxides are predominant intermediates.