Methadone N-demethylation in vitro is catalyzed by hepatic cytochrome P4502B6 (CYP2B6) and CYP3A4, but clinical disposition is often attributed to CYP3A4. This investigation tested the hypothesis that CYP2B6 is a prominent CYP isoform responsible for clinical methadone N-demethylation and clearance, using the in vivo mechanism-based CYP2B6 inhibitor ticlopidine, given orally for 4 days. A preliminary clinical investigation with the CYP3A4/5 substrate probe alfentanil established that ticlopidine did not inhibit intestinal or hepatic CYP3A4/5. Subjects received intravenous plus oral (deuterium-labeled) racemic methadone before and after ticlopidine. Ticlopidine significantly and stereoselectively (S > R) inhibited methadone N-demethylation, decreasing plasma metabolite/methadone area under the curve ratios and metabolite formation clearances. Ticlopidine also significantly increased the dose-adjusted plasma area under the curve for R- and S-methadone by 20% and 60%, respectively, after both intravenous and oral dosing. CYP2B6 inhibition reduces methadone N-demethylation and clearance, and alters methadone concentrations, demonstrating an important role for CYP2B6 in clinical methadone disposition.