Metabolism of the new nonsteroidal antiinflammatory drug aceclofenac ([2-(2',6'-dichlorophenylamino)phenyl]acetoxyacetic acid) was investigated both in the in vitro hepatic human models and in vivo. Aceclofenac is metabolized in human hepatocytes and human microsomes to form [2-(2',6'-dichloro-4'-hydroxy- phenylamino)phenyl]acetoxyacetic acid as the major metabolite, which is then further conjugated. Minor metabolites were [2-(2',6'-dichlorophenylamino)-5-hydroxyphenyl]acetoxyacetic acid and [2-(2',6'-dichlorophenylamino)phenyl]acetic acid, as well as the hydroxylated derivatives [2-(2',6'-dichloro-4'- hydroxyphenylamino)phenyl]acetic acid and [2-(2',6'-dichlorophenylamino)- 5-hydroxyphenyl]acetic acid. After oral administration to human volunteers (100 mg, single dose), aceclofenac reached a Cmax value of 7.6 +/- 1.3 micrograms/ml and a tmax of 2.6 +/- 1.8. The same metabolites as those detected in cell culture or microsome incubations were found in 12-hr urine after an oral administration of 100 mg aceclofenac to human volunteers. Cytochrome 2C9 is the enzyme responsible for the hydroxylation at position 4'. This could be demonstrated by: 1) selective inhibition by sulfaphenazole; 2) correlation between the formation of the hydroxylated metabolite and tolbutamide hydroxylase activity; and 3) formation of this metabolite only when incubated with microsomes obtained from cells expressing human cytochrome 2C9. However, no conclusive information could be obtained concerning the cytochrome catalyzing the hydroxylation at position 5. The comparison between human microsomes and human hepatocytes metabolism on one hand, and human in vivo metabolism on the other, supports human hepatocytes in primary culture as the model that best anticipated the metabolism of the drug in vivo.