Extracellular recordings were made from single neurons in the myenteric plexus of the guinea-pig ileum in vitro. Tissue was removed from morphine-naive guinea pigs and maintained in vitro for up to 24 hr; the firing rate of the neurons was reduced by opiate agonists and unaffected by opiate antagonists. Tissues were also removed from animals which had been pretreated with morphine during 3 days and placed into an in vitro solution which contained morphine. An increase in the concentration of morphine did not inhibit neuronal firing and naloxone caused a pronounced excitation of such cells. Cross-tolerance among different opiate agonists was apparent. In a third experiment, tissues were removed from morphine-naive guinea pigs and incubated in vitro for 24 hr in a solution which contained an opiate agonist. An increase in the concentration of agonist did not inhibit cell firing and opiate antagonists caused marked excitations of neurons incubated with morphine. Incubation with opiate agonists induced a much reduced sensitivity to the inhibitory effect of morphine on cell firing (tolerance) and also sensitized the cells to a marked excitatory effect of opiate antagonists (dependence) which was similar to the changes induced by in vivo opiate administration. The changes induced by morphine during 24 hr in vitro were not affected by the concomitant presence of sufficient lidocaine to prevent neuronal activity.