Dependence can be induced and measured in vitro by using guinea-pig ileum. After a brief exposure to opioids, tissues from untreated animals, show a strong naloxone-induced contracture indicating that the cellular mechanisms of dependence may occur very rapidly following occupation of receptors and that these mechanisms operate within the myenteric plexus. Several observations indicate that acethilcoline (Ach) is involved in pharmacological effects of morphine and it is one of neurotransmitters widely involved in the expression of opioid withdrawal. This is confirmed by data showing that atropine, a well known muscarinic receptor, is able to block the symptoms of opiate withdrawal. Five subtypes muscarinic receptors, M1-M5, have been identified by pharmacological studies. The availability of agonists and antagonists for M1-M5, provides powerful tools that can be used to determine the roles of these receptor types in mediating some of physiological and pharmacological effects of muscarinic receptors in the central nervous system (CNS). The experiments described here were undertaken to provide insight into the role of M1-M5 muscarinic receptors subtypes in mediating opioid withdrawal. Therefore, the effects exerted by M1-M5 muscarinic receptors antagonists on the acute opiate withdrawal induced by opioids were investigated in vitro. Following a 4 min in vitro exposure to morphine or DAGO or U50-488H the guinea-pig isolated ileum exhibited a strong contracture after the addition of naloxone. The addition of pirenzepine, the M1 muscarinic receptor antagonist (1x10-8-5x10-8-1x10-7 M) 10 min before each opioid agonist produced a concentration-dependent reduction of the opioid withdrawal. Very similar results were obtained with metoctramine (M2 muscarinic receptor antagonist) with 4-DAMP (M3 muscarinic receptor antagonist) with himbacine (M4 muscarinic receptor antagonist) and PD 102807 (M5 muscarinic receptor antagonist). The results of our experiments indicate that M1-M5 muscarinic receptors antagonists are able to reduce opioid withdrawal in vitro thus confirming an important functional interaction between the muscarinic system and opioid withdrawal.