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The objective of this investigation was to screen for potential endogenous substrates for CYP2D6. Using recombinant CYP2D6, together with hepatic microsomes from CYP2D6-transgenic mice, human liver microsomes, and a specific anti-CYP2D6 monoclonal antibody, it was ascertained that CYP2D6 does not significantly metabolize the endogenous phenylethylamines 2-phenylethylamine, octopamine, synephrine, 3-methoxy-p-tyramine, 4-methoxy-m-tyramine, metanephrine, and normetanephrine, nor the indolethylamines tryptamine, serotonin, 6-methoxytryptamine, and melatonin, nor the β-carbolines harman, norharman and tryptoline. However, the indolethylamines 5-methoxy-N,N-dimethyltryptamine (5-MDMT) and pinoline (6-methoxy-1,2,3,4-tetrahydro-β-carboline) showed relatively high affinity for CYP2D6 in a spectral binding assay (Ks 28 ± 5, and 0.5 ± 0.3 μm (mean ± SEM), respectively) and were O-demethylated only by CYP2D6 in a panel of 15 recombinant common human P450s. Pinoline and 5-MDMT O-demethylase activities were 35- and 11-fold greater in liver microsomes from CYP2D6-humanized mice, respectively, than those in liver microsomes from control mice. Moreover, the increased activities were completely inhibited by an anti-CYP2D6 monoclonal antibody. Kinetic analysis with recombinant CYP2D6 gave Km and kcat values for 5-MDMT and pinoline O-demethylations of 12 ± 1 μm and 65 ± 1 min−1 and 1.8 ± 0.3 μm and 26 ± 1 min−1, respectively. These two substrates can be added to 5-methoxytryptamine, which we have recently reported to be an endogenous CYP2D6 substrate. CYP2D6 is therefore a relatively highly specific, high-affinity, high-capacity 5-methoxyindolethylamine O-demethylase. Polymorphic cytochrome CYP2D6 may therefore exert an influence on mood and behavior by the O-demethylation of these 5-methoxyindolethylamines found in the brain and pineal gland. These processes may also impact on mental and neurological health. The findings may open new vistas for the determination of CYP2D6 phenotype.