Increased global DNA methylation in the blood of patients chronically exposed to opioids had been interpreted as an indication of an epigenetic action of this drug class.To strengthen the causality, human MCF7 cells were cultured in media with the addition of several known or potential modulators of DNA methylation including methadone.Following 3 days of incubation with several different known or potential epigenetic modulators, global DNA methylation, quantified at LINE-1 CpG islands, showed a large variability across all treatments ranging from 27.8 to 63%. Based on distribution analysis of the global methylation of human DNA exposed to various potential modulators, present in vitro experiments showed that treatment with the opioid methadone was associated with an increased probability of hypermethylation.This strengthens the evidence that opioids interfere with mechanisms of classical epigenetics.