The abuse-related behavioral effects produced by nitrous oxide (N2O) gas have been suggested as being unique compared with other abused inhalants. The drug discrimination paradigm in animals can be used to study subjective effects of drugs in humans and to test this hypothesis. The goals of the present experiment were to establish N2O discrimination in mice and to compare its discriminative stimulus effects with those of abused volatile vapors and vapor anesthetics. Sixteen B6SJLF1/J mice were trained to discriminate between 10 min of exposure to 60% N2O+40% oxygen (O2) and 10 min of exposure to 100% O2. The time course of N2O discrimination was examined, followed by cross-substitution testing with abused vapors, volatile anesthetics, ethanol, D-amphetamine, and 2-butanol. Mice acquired the ability to discriminate between N2O and O2 in 40 days. N2O fully substituted for 10 min of exposure to 60% N2O in a concentration-dependent manner. Full substitution required 7 min of 60% N2O exposure, but the offset of stimulus effects following the cessation of exposure was more rapid. The aromatic hydrocarbon toluene almost fully substituted for N2O. 1,1,1-Trichloroethane, methoxyflurane, isoflurane, and ethanol showed lesser degrees of substitution. D-amphetamine and the odorant 2-butanol did not substitute for N2O. Given the varying degrees of incomplete substitution by test compounds, the discriminative stimulus properties of N2O and, perhaps, its subjective effects in humans are probably not unique. As none of the inhalants tested fully mimicked N2O, its overall effects may include one or more novel stimulus components.