Previous reports have shown that rat brain microsomes are capable of metabolizing tricyclic antidepressants such as imipramine. Subsequent studies have shown that the protein products of several clones isolated from rat brain cDNA libraries are capable of metabolizing imipramine to both its active metabolite, desipramine, and its inactive hydroxylated metabolites. We report here the overexpression and partial purification of P450 2D18 using the baculovirus expression system and the incorporation of a C-terminal [His]₄tag. P450 2D18 was partially purified to a specific content of 4.8 nmol/mg protein and shown to be electrophoretically pure. The apparentK_Mvalues for P450 2D18 toward imipramine and desipramine were 374 and 314 μM, respectively. While apparentK_Mvalues were similar, P450 2D18 was shown to have a fivefold increasedV_max(2.2 nmol/min/nmol P450) for imipramine compared to desipramine (0.44 nmol/min/nmol P450), suggesting a primary involvement in the activation of imipramine to desipramine. We also examined the effect of theCYP2D6 inhibitor quinidine, theCYP3A inhibitor ketoconazole, and the dopamine reuptake inhibitor GBR-12935 for their ability to inhibit P450 2D18-mediated metabolism of imipramine. These results, when compared with previous studies using rat brain microsomes, suggest that P450 2D18 may play an important role in the conversion of imipramine to its active metabolite desipramine in the rat brain.