Nitrous oxide produces behavioral effects, the underlying mechanism of which is not known. In the mouse staircase test, exposure to nitrous oxide caused a reduction in rearing activity, an effect similar to that produced by benzodiazepines in this paradigm, when its opioid action on locomotion is blocked by naloxone. In this study, we tested whether effects of nitrous oxide might be mediated by benzodiazepine receptors, using chlordiazepoxide as a control. The abilities of nitrous oxide and chlordiazepoxide to reduce rearing were significantly attenuated in mice pretreated with the benzodiazepine receptor blocker flumazenil or rendered tolerant to benzodiazepines. These findings suggest an involvement of benzodiazepine receptors in mediation of certain behavioral effects of nitrous oxide.