The long-acting opioid methadone displays considerable unexplained interindividual pharmacokinetic variability. Methadone metabolism clinically occurs primarily by N-demethylation to 2-ethyl-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), catalyzed predominantly by CYP2B6. Retrospective studies suggest that the common allele variant CYP2B6*6 may influence methadone plasma concentrations. The catalytic activity of CYP2B6.6, encoded by CYP2B6*6, is highly substrate-dependent. This investigation compared methadone N-demethylation by CYP2B6.6 with that by wild-type CYP2B6.1. Methadone enantiomer and racemate N-demethylation by recombinant-expressed CYP2B6.6 and CYP2B6.1 was determined. At substrate concentrations (0.25-2 µM) approximating plasma concentrations occurring clinically, rates of methadone enantiomer N-demethylation by CYP2B6.6, incubated individually or as the racemate, were one-third to one-fourth those by CYP2B6.1. For methadone individual enantiomers and metabolism by CYP2B6.6 compared with CYP2B6.1, Vmax was diminished, Ks was greater and the in vitro intrinsic clearance was diminished 5- to 6-fold. The intrinsic clearance for R- and S-EDDP formation from racemic methadone was diminished approximately 6-fold and 3-fold for R- and S-methadone, respectively. Both CYP2B6.6 and CYP2B6.1 showed similar stereoselectivity (S>R-methadone). Human liver microsomes with diminished CYP2B6 content due to a CYP2B6*6 allele had lower rates of methadone N-demethylation. Results show that methadone N-demethylation catalyzed by CYP2B6.6, the CYP2B6 variant encoded by the CYP2B6*6 polymorphism, is catalytically deficient compared with wild-type CYP2B6.1. Diminished methadone N-demethylation by CYP2B6.6 may provide a mechanistic explanation for clinical observations of altered methadone disposition in individuals carrying the CYP2B6*6 polymorphism.