We have previously shown that the antinociceptive effect of nitrous oxide (N 2 O) in the rat hot plate test is sensitive to antagonism by antisera against the endogenous opioid peptide β-endorphin. Moreover, N 2 O-induced antinociception is reduced by inhibition of nitric oxide (NO) production in the brain. To test the hypothesis that N 2 O might stimulate an NO-dependent neuronal release of β-endorphin, we conducted a ventricular-cisternal perfusion with artificial cerebrospinal fluid (aCSF) in urethane-anesthetized Sprague Dawley rats. Ten-min fractions of aCSF perfusate were collected from separate groups of room air-exposed rats, N 2 O-exposed rats, and L-NAME-pretreated, N 2 Oexposed rats; they were then analyzed for their content of NO metabolites and β-endorphin. Compared to room air control, exposure to 70% N 2 O increased perfusate levels of the NO metabolites nitrite and nitrate as well as β-endorphin. Pretreatment of rats with L-N G -nitro arginine methyl ester, an inhibitor of NO synthase, prevented the N 2 O-induced increases in nitrite, nitrate and β-endorphin. These findings demonstrate in an in vivo rat model that N 2 O may stimulate an NO-dependent neuronal release of β-endorphin.