Tienilic acid (TA) is metabolized by liver microsomes from phenobarbital-treated rats in the presence of NADPH with the major formation of 5-hydroxytienilic acid (5-OHTA) which is derived from the regioselective hydroxylation of the thiophene ring of TA. During this in vitro metabolism of TA, reactive electrophilic intermediates which bind irreversibly to microsomal proteins are formed. 5-Hydroxylation of TA and activation of TA to reactive metabolites which covalently bind to proteins both required intact microsomes, NADPH and O2 and are inhibited by metyrapone and SKF 525A, indicating that they are dependent on monooxygenases using cytochromes P-450. Microsomal oxidation of an isomer of tienilic acid (TAI) bearing the aroyl substituent on position 3 (instead of 2) of the thiophene ring also leads to reactive intermediates able to bind covalently to microsomal proteins. Covalent binding of TAI, as that of TA, depends on cytochrome P-450-dependent monooxygenases and is almost completely inhibited in the presence of sulfur containing nucleophiles such as glutathione, cysteine or cyteamine. These results show that 5-OHTA, which has been reported as the major metabolite of TA in vivo in humans, is formed by liver microsomes by a cytochrome P-450-dependent reaction. They also show that two thiophene derivatives, TA and TAI, bind to microsomal proteins after in vitro metabolic activation, TAI giving a much higher level of covalent binding than TA (about 5-fold higher) and a much higher covalent binding: stable metabolites ratio (4 instead of 0.5).