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To characterize the nitrous oxide-induced inhibition of the enzyme, methionine synthetase, we measured enzyme inactivation as a function of nitrous oxide concentration and exposure time. Mice exposed to 0.8 atm nitrous oxide exhibited more than a 50 per cent decrease in liver methionine synthetase activity within 30 min, and activity dropped to 5–25 per cent of the original value after a 4-hour exposure. Although 4-hour exposures to low nitrous oxide partial pressures (less than 0.05 atm) did not significantly alter methionine synthetase activity, higher concentrations of nitrous oxide caused a progressive inhibition over this time period. Continuous exposure to trace levels of nitrous oxide (approximately 1100 ppm) for eight to 22 days produced a small but significant reduction in liver and brain methionine synthetase activity. Methionine synthetase activity returned to control levels two to four days following inactivation. Other anesthetics (xenon, halothane, isoflurane, enflurane) did not produce inactivation.