Some experts suggest that sedation of laboratory rodents with isoflurane before euthanasia with carbon dioxide (CO2) is a humane alternative to euthanasia with CO2 alone, but little research has compared aversion to these agents. Albino rats were tested in a light/dark box where they had the choice between remaining in a dark compartment filling with isoflurane or CO2, or escaping to a lit compartment. Experiment 1 validated the procedure by confirming that rats responded to agent and light intensity. In Experiment 2, 9/16 and 0/16 rats remained in the dark compartment until recumbent when initially exposed to isoflurane and CO2, respectively. In Experiment 3, more rats remained in the dark compartment until recumbent during initial (10/16) versus re-exposure (1/16) to isoflurane. These results indicate that initial exposure to CO2 is more aversive than isoflurane, and that re-exposure to isoflurane is more aversive than initial exposure. We conclude that sedation with isoflurane is a refinement over euthanasia with CO2 alone for rats that have not been previously exposed to inhalant anaesthetics.