The formation of three oxidative metabolites of imipramine, N-desmethylimipramine (desipramine), 2-hydroxyimipramine, and 10-hydroxyimipramine was studied in microsomes of an extensive metabolizer liver (KDL 26) and of a poor metabolizer liver (KDL 31) and in a homogenate of COS-1 cells in which the P450IID6 complementary deoxyribonucleic acid had been expressed. The following data support the role of P450IID6 in the 2-hydroxylation of imipramine: (1) The formation of 2-hydroxyimipramine was reduced to less than 20% of the control value when microsomes were incubated with serum containing inhibitory antibodies against P450IID6 (anti-LKM1), whereas no effect was seen with regard to formation of desipramine and 10-hydroxyimipramine, (2) quinidine and levomepromazine were potent competitive inhibitors of 2-hydroxylation of imipramine (ki approximately 70 nmol/L, and ki approximately 1 mumol/L, respectively) but had no effect on N-demethylation and 10-hydroxylation, and (3) in the COS-1 cell, homogenate, 10-hydroxyimipramine, 2-hydroxyimipramine, and desipramine were formed at rates of 48, 164, and 256 pmol per hour per milligram of homogenate protein, respectively. The P450 isozymes that are responsible for N-demethylation and 10-hydroxylation of imipramine have not yet been identified.