The psychotropic β-carboline alkaloids, showing high affinity for 5-hydroxytryptamine (5-HT), dopamine, benzodiazepine and imidazoline receptors and the stimulation of locus coeruleus neurons, are formed endogenously from tryptophan-derived indolealkylamines through the Pictet-Spengler condensation with aldehydes in both plants and mammals. Cytochromes P450 (CYP) 1A1 (18.5), 1A2 (20) and 2D6 (100) catalyzed the O-demethylation of harmaline, and CYP1A1 (98.5), CYP1A2 (35), CYP2C9 (16), CYP2C19 (30) and CYP2D6 (115) that of harmine (relative activities). The dehydrogenation/aromatization of harmaline to harmine was not carried out by aromatase (CYP19), CYP1A2, CYP2C9, CYP2D6, CYP3A4, pooled recombinant P450s, or by human liver microsomes (HLM). Kinetic parameters were calculated for the Odemethylations mediated by each isozyme and by pooled HLM. Kcat (min-1) and Km (µM) values for harmaline were CYP1A1 (10.8, 11.8), CYP1A2 (12.3, 13.3), CYP2C9 (5.3, 175), CYP2C19 (10.3, 160), and CYP2D6 (39.9, 1.4), and for harmine were CYP1A1 (45.2, 52.2), CYP1A2 (9.2, 14.7), CYP2C9 (11.9, 117), CYP2C19 (21.4, 121), and CYP2D6 (29.7, 7.4). Inhibition studies using monoclonal antibodies confirmed that CYP1A2 and CYP2D6 were the major isozymes contributing to both harmaline (20% and 50% respectively) and harmine (20% and 30%) O-demethylations in pooled HLM. The turnover numbers for CYP2D6 are amongst the highest ever reported for a CYP2D6 substrate. Finally, CYP2D6-transgenic mice were found to have increased harmaline and harmine O-demethylase activities as compared to wild-type mice. These findings suggest a role for polymorphic CYP2D6 in the pharmacology and toxicology of harmine and harmaline.