The fate of phenacetin and some of tis metabolites have been examined in isolated rat hepatocytes. The overall pattern of metabolism was similar to that found in vivo by others. The major metabolites of phenacetin were paracetamol, free and conjugated, and phenetidine, and about 10% was lost. No N-hydroxyphenacetin was found, but experiments with N-hydroxyphenacetin as substrate showed that at low concentration (as might be formed from phenacetin) it disappeared very rapidly from cell suspensions. N-hydroxyphenacetin was metabolized to its conjugates, and to paracetamol, phenacetin and phenetidine, with a large proportion unaccounted for. With all substrates, increasing concentration resulted in a decreased percentage being metabolized, indicating that the metabolic pathways were saturable. Relatively more phenetidine was found at high phenacetin concentrations, however, apparently because phenetidine is an intermediate metabolite whose own elimination was slowed relatively more than its formation. N-hydroxylated arylamines were toxic to hepatocytes in a dose-dependent manner, suggesting that these cell suspensions could be used to test for hepatotoxicity.