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Groups of rats were exposed to air or 80 per cent nitrous oxide for 30 min or 18 h, following which the brainstem opiate receptor density and the apparent affinity of these receptors to the radiolabeled agonist, 3H-dihydromorphine, were assayed. Thirty-minute exposure to nitrous oxide did not change opiate receptor characteristics, immediately or 17.5 h later. However, prolonged exposure to nitrous oxide (18 h) decreased the brainstem opiate receptor density approximately 20 per cent, without a change in apparent receptor affinity. These results support the view that nitrous-oxide-induced analgesia results from release of endogenous opiate-like substances. Continued presence of these substances in turn results in a decrease in opiate receptor density and may account for the development of tolerance to the analgesic action of nitrous oxide.