Nitrous oxide (N2O) exposure has been associated with neurotoxicity, especially peripheral neuropathy, in both humans and animals. The effects of this anesthetic gas on the central nervous system (CNS) and spontaneous behavior, however, have yet to be delineated. Timed-pregnant Sprague-Dawley rats were exposed to 75% N2O/25% O2 on gestational days 14 and 15 or day 15 only for eight hours per day. The offspring were tested at one and five months of age; their spontaneous behavior in a novel environment was recorded in the residential maze and using time-lapse photography. The results indicated that in utero exposure to N2O permanently altered the spontaneous motor output of the CNS. This effect was most prominent in 5 month old animals, and females were affected more than males. Exposures on gestational days 14 and 15 produced an effect that was not only greater but also qualitatively different than that produced by exposure on day 15 only. The two-day exposure induced hyperactivity in both sexes, whereas the one-day exposure induced hyperactivity in the males and slight hypoactivity in the females. These behavioral changes were not accompanied by physical abnormalities but nonetheless were lasting effects in need of further consideration.