Experiments were conducted on three different human liver samples to identify the cytochrome P450 isozyme which is involved in the biotransformation of the class III antiarrhythmic agent, amiodarone, into its major metabolite, desethylamiodarone (DEA). The classic P450 inhibitors, SKF 525A, metyrapone, and carbon monoxide provided a significant reduction in the in vitro formation of DEA by human hepatic microsomes. Amiodarone N-deethylase activities expressed by intrinsic clearance values were similar in all the livers used, although two livers were genotyped as extensive and one as a poor metabolizer for the cytochrome P450 CYP2D6 gene. DEA production was strongly inhibited (more than 80%) by the anti-P450 3A4 antibody, but not by anti-LKM1-positive serum. It seems therefore that the P450 3A subfamily is certainly implicated in human hepatic amiodarone N-deethylation.