In order to investigate the glucuronidation of 2-ethylhexanoic acid (2-EHA), a metabolite of the plasticizer di-(2-ethylhexyl) adipate, by liver microsomes of several mammalian species including man, a gas chromatography method for the quantification of the corresponding glucuronides was developed. The variation coefficients for intra- and interassay repeatability were less than 3 and 7%, respectively. The rat liver UDP-glucuronosyl-transferase (UGT) presented similar Km and Vmax toward the two enantiomers. The glucuronidation of the racemate 2-EHA or its enantiomers was strongly increased up to six times by treatment of the rats with phenobarbital and, to a lesser extent, by 3-methylcholanthrene. In contrast, the treatment of the rats clofibrate did not modify the activity. The induction was not stereoselective. The Gunn rats, which present a genetic defect in the bilirubin UGT isoforms, were able to glucuronidate the drug as well as the congenic strain. Moreover, the UGT-2B1 isoform, stably expressed in V79 cells, glucuronidated 2-EHA in an appreciable amount. Interspecies comparison indicated that the most active glucuronidation of 2-EHA occurred in the dog and the rat. The lowest activities were observed in the man and the rabbit. In all species considered, except rabbit and guinea pig which glucuronidated the R isomer faster, the R and S enantiomers were glucuronidated to a similar extent. The glucuronidation activity toward compounds chemically related to 2-EHA increased as a function of molecular weight, but was not affected by the position of the methyl or the ethyl moiety on the hydrocarbon chain. A correlation between the glucuronidation rate of 2-EHA and analogs and the activity of PCoA oxidase was observed.