Dependence can be induced and measured in vitro by using guinea-pig ileum. Tissues from untreated animals, after a brief exposure to opioids, 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. The characteristics of dependence development and the precipitation of withdrawal by naloxone in the guinea-pig ileum are very similar to those of acute dependence in experimental animals and man. Several observations indicate that brain serotoninergic system has been widely implicated in many of the pharmacological effects of opioids. Although the role of serotonin in the development of dependence has been previously reported the effects exerted by chronic treatment of p-Chlorophenylalanine (p-CPA), an inhibitor of tryptophan 5-hydroxylation which is the rate limiting step in the synthesis of serotonin, on the acute opiate withdrawal was investigated in vitro and whether p-CPA interferences on opiate withdrawal take place through μ and/or κ opioid receptors. Following a 4 min in vitro exposure to the opioid agonist (Morphine, DAGO or U50-488H), the guinea-pig isolated ileum exhibited a strong contracture after the addition of naloxone (80% of contraction vs. acetylcholine control). By contrast, ilea from guinea-pigs treated for 6 days with p-CPA (10 mg/Kg/i.p.), following a 4 min in vitro exposure to the opioid agonist, did not show any contracture after the addition of naloxone (0% of contraction vs. acetylcholine control). The results of the present study confirm an important functional interaction between the serotoninergic system and opioid system.