Isopryopylbenzene-degrading bacteria, including Pseudomonas putida RE204, transform benzothiophene to a mixture of compounds. Induced strain RE204 and a number of its Tn5 mutant derivatives were used to accumulate these compounds and their precursors from benzothiophene. These metabolites were subsequently identified by (1)H and (13)H nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry. When strain RE204 was incubated with benzothiophene, it produced a bright yellow compound, identified as trans-4-(3-hydroxy-2-thienyl)-2-oxobut-3-enoate, formed by the rearrangement of cis-4-(3-deto-2,3-dihydrothienyl)-2-hydroxybuta-2,4-dienoate, the product of 3-isopropylcatechol-2,3-dioxygenase-catalyzed ring cleavage of 4,5-dihydroxybenzothiophene, as well as 2-mercaptophenylglyoxalate and 2'-mercaptomandelaldehyde. These data indicate that oxidation of benzothiophene by strain RE204 is initiated at either ring.