Withdrawal syndrome after the cessation of μ-opioid receptor agonists remains an obstacle in the clinical treatment of pain. There is limited information available on the mechanisms that underlie the expression of the withdrawal signs of opioids, and especially regarding the involvement of μ-opioid receptor subtypes and the location of the responsible opioid receptors. Therefore, the present study was designed to determine the mechanism of the expression of withdrawal signs in μ-opioid receptor agonist-dependent mice. Morphine-, oxycodone- and fentanyl-dependent mice showed a marked loss of body-weight and other signs of withdrawal after a naloxone challenge. Interestingly, the phenotype of the withdrawal signs for morphine and oxycodone was different from that of fentanyl. Furthermore, pretreatment with naloxonazine (so-called μ1-opioid receptor antagonist), did not significantly alter the withdrawal signs precipitated by naloxone in these μ-opioid receptor agonist-dependent mice, whereas the peripherally limited opioid receptor antagonist naloxone methiodide significantly increased the loss of body-weight accompanied by diarrhea, indicating that a peripheral naloxonazine-insensitive site for opioid receptors, as an adaptation mechanism, plays an important role in the expression of at least the loss of body-weight. On the other hand, i.c.v. treatment with naloxone methiodide potently induced jumping behavior and trembling in morphine-dependent mice. These results indicate that the prolonged activation of supraspinal μ-opioid receptors plays a role in most of the physical dependence induced by μ-opioid receptor agonists in mice. Thus, the withdrawal symptoms observed after the cessation of μ-opioid receptor agonists are distinctly regulated though supraspinal and peripheral naloxonazine-insensitive sites of μ-opioid receptors.