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Carbon dioxide (CO 2 ) is one of the most commonly used gas euthanasia agents in mice, despite reports of aversion and nociception. Inert gases such as nitrogen (N 2 ) may be a viable alternative to carbon dioxide. Here we compared behavioural and electrophysiological reactions to CO 2  or N 2  at either slow fill or rapid fill in C57Bl/6 mice undergoing gas euthanasia. We found that mice euthanised with CO 2  increased locomotor activity compared to baseline, whereas mice exposed to N 2  decreased locomotion. Furthermore, mice exposed to CO 2  showed significantly more vertical jumps and freezing episodes than mice exposed to N 2 . We further found that CO 2  exposure resulted in increased theta:delta of the EEG, a measure of excitation, whereas the N 2  decreased theta:delta. Differences in responses were not oxygen-concentration dependent. Taken together, these results demonstrate that CO 2  increases both behavioural and electrophysiological excitation as well as producing a fear response, whereas N 2  reduces behavioural activity and central neurological depression and may be less aversive although still produces a fear response. Further studies are required to evaluate N 2  as a suitable euthanasia agent for mice.

Nitrogen gas produces less behavioural and neurophysiological excitation than carbon dioxide in mice undergoing euthanasia