Methadone is widely used as a maintenance treatment for opiate addiction. Methadone plasma levels vary widely for a given dose, so contributing to interindividual variability in response to methadone maintenance treatment. Until recently, the relative in vivo involvement of various cytochrome P450 (CYP) isoforms in methadone pharmacokinetics had been unclear. A recent large-scale pharmacogenetic study with patients in methadone maintenance treatment has now demonstrated that CYP3A4 and CYP2B6 are the major cytochrome P450 isoforms with a major involvement in methadone metabolism, while CYP2D6 only contributes to a minor extent. In addition, P-glycoprotein, a transmembrane efflux protein, is also involved in methadone kinetics.