Euthanasia is a necessary component in research and must be conducted humanely. Currently, regulated CO2 exposure in conscious rats is acceptable, but data are divided on whether CO2 alone is more distressing than anesthesia prior to CO2. To evaluate distress in rats, we compared physiologic responses to CO2 euthanasia with and without isoflurane preanesthesia. Male Sprague–Dawley rats were implanted with telemetry devices to measure mean arterial pressure (MAP), heart rate (HR), and blood glucose. Animals recovered for 2 wk and were then exposed to either 5% isoflurane (n = 6) or 100% CO2(n = 7; calculated 30% chamber volume/min displacement) in their home cages to induce loss of consciousness. Euthanasia was then completed with CO2 in both groups. MAP and HR increased when the gas delivery lids were placed on the home cages of both groups. Both MAP and HR gradually decreased with isoflurane exposure. MAP increased and HR decreased with CO2 exposure. Glucose levels remained stable throughout the procedure, except for a small drop in conscious animals initially exposed to 100% CO2. These data suggest that both gases affect the measured parameters in a similar manner, andthat environmental factors, such as gas delivery lid placement, also change these measurements.