The American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals: 2020 Edition [1] provides clarification concerning use of CO2 exposure as a method of euthanasia for laboratory rodents. As such, the ARC has developed the following procedural requirements in order to conform to these guidelines.

  1. The practice of immersion, where conscious animals are placed directly into a container prefilled with 100% CO2, is not acceptable per the aforementioned AVMA Guidelines. Carbon dioxide exposure using a gradual fill method is considered less likely to cause pain because analgesia is induced at approximately 7.5%, and anesthesia is induced at 30%, with death due to nervous system depression occurring with prolonged concentrations of 30% and higher.
  2. Therefore, a gradual filling method must be used and animals must be observed continuously for the duration of the procedure.
      1. The only acceptable condition where an animal may be placed directly into a high concentration of CO2 is when the animal has been rendered unconscious first, such as with inhaled or injectable anesthetics.
      2. The recommended fill rate (displacement) of the euthanasia chamber is between 30% and 70% of the chamber volume per minute. CO2 must be supplied from either a gas cylinder or a facility CO2 gas distribution system equipped with an appropriate pressure-reducing regulator and flow meter combination (or equivalent) to permit precise regulation of gas flow to the chamber.
      3. CO2 delivery via dry ice is NOT an acceptable method.

    Example Flow Rates for 60% Volume Displacement per Minute [2]

    Cage TypeCage Size (Wx Lx H)Flow Rate
    Mouse6.5" x 12" x 5.5"

    4.2 liters/min

    Rat9" x 17.5" x 8"

    12.4 liters/min

  3. Using this gradual fill method, CO2 flow should be maintained for at least 1 minute after respiratory arrest for adult rodents and a confirmatory method of euthanasia appropriate to the species must be used.
    1. For mice, acceptable confirmatory methods after CO2 exposure include:
      1. decapitation,
      2. cervical dislocation,
      3. thoracotomy,
      4. immediate harvest of critical organs, and
      5. observation for lack of heartbeat and respiration, with graying of mucus membranes, for at least ten (10) minutes.
    2. For rats, a physical method must be used to confirm death following CO2 exposure, unless the Euthanex Smartbox chamber is used for euthanasia of adult rats. Acceptable physical methods include:
      1. decapitation,
      2. cervical dislocation (for rats weighing less than 200g only),
      3. thoracotomy, and
      4. immediate harvest of critical organs.

  4. Since rodent pups are much less sensitive than adults to the effects of CO2, either a secondary, physical method of euthanasia after CO2 exposure OR an alternative method of euthanasia must be used. Refer to the  ARC Policy on Euthanasia of Fetuses and Neonates – Mice and Rats for more information.

  5. It is preferable that animals are euthanized in their home cage to minimize stress (for example, if two mice out of five are to be euthanized, move three remaining mice to a clean cage and euthanize the two mice in the home cage bottom). If multiple rodents are to be combined in the same container for euthanasia (for example, two singly housed females), they must be of the same species. Do not over-crowd the cage; apply the same cage limits as for rodents under your care. Do not combine male mice, as they will fight.

  6. The euthanasia chamber should be cleaned between uses. This will also purge the chamber of residual CO2 between cages.

It cannot be overstated that death must be confirmed before disposal of animal remains. For training on confirmatory methods, contact


  1. AAVMA Guidelines for the Euthanasia of Animals: 2020 Edition, p 5 and pp 28-31
  2. DLAM CO2 euthanasia stations will have posted signage to instruct users to deliver 60% fill rate. For lab-maintained stations, use the following equation to determine CO2 flow for a 30-70% per minute replacement rate of room air by CO2:
Image of equation to determine CO2 flow for a 30-70% per minute replacement rate of room air by CO2

Where height, width and length are in inches and 61 is the conversion factor for in3 to liters.

From Guidelines for Euthanasia of Rodents using Carbon Dioxide NIH Office of Intramural Research, Office of Animal Care and Use. Accessed 03.28.2023

Approved 3/24/14, Revised 9/14/20, 4/10/23, 7/10/23