Polymorphism at the cytochrome P450 2D6 (CYP2D6) locus is one of the most widely known causes of pharmacogenetic variability in humans. Our goal is to investigate the intrinsic enzymatic differences that exist among active CYP2D6 isoforms to test the hypothesis that these enzymatic differences are substrate-dependent. Active CYP2D6.1, 2, 10, and 17 holoenzymes were expressed in vitro and purified to a high degree of homogeneity as confirmed with SDS-polyacrylamide gel electrophoresis, CO-difference spectroscopy, and mass spectral analysis. Purified enzyme was reconstituted with lipid and cytochrome P450 reductase in a 2:1 ratio before kinetic analysis. The reaction rate for dextromethorphan (DXM) O-demethylation, DXM N-demethylation, codeine O-demethylation, and fluoxetine N-demethylation catalyzed by each of the variants was determined. The CYP2D6.10 enzyme was the most impaired, exhibiting an estimated enzyme efficiency (as V max /K m ) 50-fold lower for DXM O-demethylation and 100-fold lower for fluoxetine N-demethylation when compared with CYP2D6.1, whereas no measurable catalytic activity was observed for this variant toward codeine. The atypical DXM N-demethylation pathway catalyzed by this variant decreased only 2-fold in comparison. In the case of CYPD6.17, estimated clearances for each metabolite were decreased 6 to 33%. Likewise, the intrinsic clearance of CYP2D6.2 enzyme was consistently decreased for each reaction examined, indicating that the ultra-rapid metabolizer phenotype sometimes associated with this genotype is not a function of the underlying amino acid substitutions. Overall enzyme efficiencies for the metabolism of each substrate therefore decreased in the order of 2D6.1 Ͼ 2D6.2 Ͼ 2D6.17 Ͼ 2D6.10.Cytochrome P450 enzymes, a superfamily of heme-thiolate proteins, are found in all living organisms and are involved in the biotransformation of a diverse range of xenobiotics and endobiotics. Human P450 isoforms, which are mainly expressed in the liver, play a central role in drug metabolism. Variations in individual metabolism often result in unexpected toxicities because drug clearance is affected by a range of factors, including genetic variation, enzyme induction (activation), and inhibition of drug metabolism. Therefore, characterization of the P450 enzyme family has been of unceasing interest for the prediction and identification of drug metabolism and drug-drug interactions for discovery, development, and clinical therapy (Gonzalez and