Although methadone maintenance treatment (MMT) has long been used for opioid addiction, our knowledge on its pharmacokinetics is still limited. We aimed to investigate effects of age, gender, and various co-medications on methadone serum concentration-to-dose ratio (CDR) in a naturalistic setting. In total, 4425 routine serum methadone concentrations obtained from 1691 MMT patients in the period October 1999 to July 2017 were included. Information about doses, age, gender, and concurrent medications was available in the laboratory database at the Department of Clinical Pharmacology at St. Olav University Hospital in Trondheim, Norway. A log-linear mixed model was used when analyzing the data. Mean age was 38.4 (range, 21-78) years and 70% were men. Mean CDR was 332 (range, 7-1776) (ng/mL)/(100 mg/d). Concomitant medication with at least one out of totally 170 drugs was recorded in 26% of the samples. CDRs were significantly lower in women (- 9%; confidence interval (CI), - 13%, - 4%; p = 0.001) and with concurrent use of CYP inducers (- 36%; CI, - 44%, - 28%; p < 0.001), but higher using CYP3A4 inhibitors as co-medications (+ 36%; CI, + 10%, + 68%; p = 0.005). Our results warrant taking into consideration gender differences in methadone metabolism as well as the impact of potential drug-drug interactions to obtain an optimal therapeutic effect and avoid adverse effects in MMT. Although the clinical implications of the altered drug levels require further study, our results call for close clinical monitoring of all patients undergoing MMT, preferably along with laboratory measurements of methadone serum concentrations.