Drug interactions involving methadone and/or HIV antiretrovirals can be problematic. Mechanisms whereby antiretrovirals induce clinical methadone clearance are poorly understood. Methadone is N-demethylated to 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) by CYP2B6 and CYP3A4 in vitro, but by CYP2B6 in vivo. This investigation evaluated human hepatocytes as a model for methadone induction, and tested the hypothesis that methadone and EDDP are substrates for human drug transporters. Human hepatocyte induction by several antiretrovirals of methadone N-demethylation, and CYP2B6 and CYP3A4 transcription, protein expression and catalytic activity, and pregnane X receptor (PXR) activation were evaluated. Methadone and EDDP uptake and efflux by overexpressed transporters were also determined. Methadone N-demethylation was generally not significantly increased by the antiretrovirals. CYP2B6 mRNA and activity (bupropion N-demethylation) were induced by several antiretrovirals, as were CYP3A4 mRNA and protein expression, but only indinavir increased CYP3A activity (alfentanil dealkylation). CYP upregulation appeared related to PXR activation. Methadone was not a substrate for uptake (OCT1, OCT2, OCT3, OATP1A2, OATP1B1, OATP1B3, OATP2B1) or efflux (P-gp, BCRP) transporters. EDDP was a good substrate for P-gp, BCRP, OCT1, OCT3, OATP1A2, and OATP1B1. OATP1A2- and OCT3-mediated EDDP uptake, and BCRP-mediated EDDP efflux transport, was inhibited by several antiretrovirals. Results show that hepatocyte methadone N-demethylation resembles expressed and liver microsomal metabolism more than clinical metabolism. Compared with clinical studies, hepatocytes underreport induction of methadone metabolism by HIV drugs. Hepatocytes are not a good predictive model for clinical antiretroviral induction of methadone metabolism and not a substitute for clinical studies. EDDP is a transporter substrate, and is susceptible to transporter-mediated interactions.