The metabolism of tienilic acid, a drug containing a thiophene ring, was reinvestigated in man, rat and dog. The major urinary metabolite in man and rat was isolated and completely characterized by comparison with a synthetic compound. This metabolite derives from the hydroxylation of the thiophene ring of tienilic acid in position 5. Its isomers, 3- and 4-hydroxy-tienilic acids, were synthetized but could be detected neither in man nor in rat urine. Because of its particular behaviour toward electrophiles, 5-hydroxy-tienilic acid was found to react with diazomethane with the formation of a complex mixture of methylated products. This made difficult its measurement by a previously described GLC technique, after acidic extraction and methylation by diazomethane. A new very simple assay using HPLC and direct injection of urine is described in this paper. This assay led to a very precise and reproductible determination of tienilic acid and its hydroxylated metabolite in urine. Up to 50% of tienilic acid is excreted in man or rat urine as 5-hydroxy-tienilic acid whereas this metabolite does not appear in dog urine. These data describe the first example of metabolic hydroxylation of the thiophene ring.