For rodent species, CO2 is an acceptable form of euthanasia when used under the following guidelines:

• We do not recommend prefilling (precharging) the euthanasia chamber with CO2, since high concentrations (>70%) can cause nasal irritation and excitability. Rather, the animals should first be placed into the chamber, followed by the addition of CO2 at a low flow rate (20% of the chamber volume per minute) to complete the process. Rapid gas flows should be avoided since excessive noises ("winds") can develop and induce excitement/distress in the animals.
• Place the animals into the chamber and turn the red lever until you hear gas flowing at a low rate.
• Gas flow should be maintained for at least 1 minute after apparent clinical death (approximately 5 minutes total). A timer should be used to ensure adequate length of exposure.
• It is important to confirm that an animal is dead after removing it from the chamber. This may be accomplished by assuring lack of heartbeat or by performing cervical dislocation following CO2 narcosis or creation of a pneumothorax following CO2 narcosis.

According to the 2000 Report of the AVMA Panel on Euthanasia, "Compressed CO2 gas in cylinders is the only recommended source of carbon dioxide because the inflow to the chamber can be regulated precisely. CO2 generated by other methods such as from dry ice, fire extinguishers, or chemical means (e.g. antacids) is unacceptable."

Only one species at a time should be placed into a chamber, and the chambers must not be overcrowded. Animals must meet the minimum space requirements listed in the "GUIDE" up until the time of euthanasia. When placed into the chamber, all animals must have floor space.

Euthanasia should always be done in cohorts (live animals should not be placed in the chamber with dead animals).

Chambers should be kept clean to minimize odors that might distress animals prior to euthanasia.

Animals must not be euthanized in animal housing rooms, except under special circumstances such as during quarantine for infectious disease agents.

Neonates: Since the time period for euthanasia is substantially prolonged in neonatal rodents (rodents 13-16 days) inherent resistance to hypoxia, CO2 narcosis must be followed by decapitation after the animals lose consciousness.