Experiments were performed to induce opiate tolerance and physical dependence in vitro and assess the applicability of the procedures for studying the basic mechanisms in tolerance and dependence development. It was established that tolerance and physical dependence could be demonstrated in the excised guinea pig ileum and mouse vas deferens by direct incubation with an opiate agonist. The selectivity of the response to opiates was determined by experiments demonstrating stereospecificity, naloxone reversibility and cross-tolerance. The magnitude of tolerance and physical dependence was found to be dose, time, and agonist type-dependent. A standardized degree of tolerance and dependence were induced by using a dose of agonist sufficient to inhibit electrically stimulated contractions by 50% (IC50). In the isolated guinea pig myenteric plexus of the longitudinal muscle, incubation of the IC50 of an agonist at 37 degrees C for one hour produced a 2 to 5-fold tolerance as evidenced by the increase in its IC50. The rank order of decreasing potency in developing tolerance was morphine, normorphine and methadone. Physical dependence on the opiate was established by a contractual response to naloxone or a supersensitive response to electrical stimulus after removal of the agonist by washing. The degree of dependence developed was found to be proportional to that of tolerance. Similar incubation experiments on the mouse vas deferens with opioids also resulted in the development of tolerance and dependence. Although naloxone failed to evoke a contractual response, it enhanced dose-dependently the sensitivity of the preparation to electrical stimulus.(ABSTRACT TRUNCATED AT 250 WORDS)