This study compared euthanasia induced by rising concentrations of CO₂ in aged rats (n = 59) using different gas displacement rates. Rats were preimplanted with cardiovascular telemetry devices and had been previously used for short term safety pharmacology studies. Once fully recovered from previous studies, rats were euthanized using rising concentrations of CO₂. Three groups were exposed to gas displacement at fixed flow rates of 30%, 40%, and 50%, and 3 groups were exposed to increased flow rates at predetermined, one-minute intervals (10 to 30%, 20 to 40%, and 30 to 50%). Comparisons were based on the time taken to reach 4 critical endpoints: dyspnea, ataxia, recumbency, and death. The preimplanted telemetry devices were used to record cardiovascular parameters. Video recordings of the euthanasias were performed to allow behavioral assessment by a blind observer. The histologic effects of the different concentrations were also evaluated. No significant differences were detected between the groups in behavioral scores or histopathology. Groups of rats exposed to higher levels of CO₂ had a shorter time to loss of consciousness and death than did rats exposed to lower concentrations of CO₂. No statistically significant differences were detected in the time by which rats showed visual signs of dyspnea. Slow CO₂ displacement rates of CO2 may prolong the time necessary for euthanasia yet provide no appreciable improvement in welfare in aged rats and should therefore be avoided.