1. The aromatic 2-hydroxylation of imipramine was studied in microsomes from three human livers. The kinetics were best described by a biphasic enzyme model. The estimated values of Vmax and Km for the high affinity site ranged from 3.2 to 5.7 nmol mg-1 h-1 and from 25 to 31 microM, respectively. 2. Quinidine was a potent inhibitor of the high affinity site for the 2-hydroxylation of imipramine in microsomes from all three human livers, with apparent Ki-values ranging from 9 to 92 nM. This finding strongly suggests that the high affinity enzyme is CYP2D6, the source of the sparteine/debrisoquine oxidation polymorphism. 3. The selective serotonin reuptake inhibitors (SSRI), paroxetine, fluoxetine and norfluoxetine were potent inhibitors of the high affinity site having apparent Ki-values of 0.36, 0.92 and 0.33 microM, respectively. Three other SSRIs, citalopram, desmethylcitalopram and fluvoxamine, were less potent inhibitors of CYP2D6, with apparent Ki-values of 19, 1.3 and 3.9 microM, respectively. 4. Among 20 drugs screened, fluvoxamine was the only potent inhibitor of the N-demethylation of imipramine, with a Ki-value of 0.14 microM. 5. Neither mephenytoin, citalopram, diazepam, omeprazole or proguanil showed any inhibition of the N-demethylation of imipramine and the role of the S-mephenytoin hydroxylase for this oxidative pathway could not be confirmed.