Oxidation of 2-phenylthiophene (2PT) by rat liver microsomes, in the presence of NADPH and glutathione (GSH), led to three kinds of metabolites whose structures were established by H-1 NMR and mass spectrometry. The first ones were 2PT-S-oxide dimers formed by Diels-Alder type dimerization of 2PT-S-oxide, while the second ones were GSH adducts derived from the 1,4-Michael-type addition of GSH to 2PT-S-oxide. The third metabolites were GSH adducts resulting from a nucleophilic attack of GSH to the 4,5-epoxide of 2PT. Oxidation of 2PT by recombinant, human cytochrome P4501A1, in the presence of NADPH and GSH, also led to these three kinds of metabolites. These results provide the first evidence that cytochrome P450 may catalyze the oxidation of thiophene compounds with the simultaneous formation of two reactive intermediates, a thiophene-S-oxide and a thiophene epoxide. (c) 2005 Elsevier Inc. All rights reserved.