This study examined naloxone-precipitated withdrawal symptoms from 24 to 168 h after pretreatment with a single 30-mg i.m. dose of methadone in 6 male subjects who were experienced users of opioid drugs but were not currently dependent. The study showed that acute physical dependence signs and symptoms could be reliably precipitated with a small dose of naloxone (0.75 mg/70 kg i.m.) for as long as 96 h (4 days) after a single dose of methadone. The intensity of symptoms at 24 h post-methadone was similar to that observed at 96 h; no precipitated withdrawal effects were observed at 168 h (7 days) after methadone administration. The magnitude of precipitated withdrawal effects at 96 h was not attenuated by the administration of a prior naloxone challenge at 24 h post-methadone. Agonist effects (pupillary constriction; subjective effects) were detectable at 24 h but not at 96 h post-methadone. The results suggest that methadone engenders long-lasting physical dependence effects that can be detected beyond the dissipation of acute agonist effects. Methadone pretreatments may provide a convenient mechanism for the production and examination of long-term mu-opiate receptor physical dependence.