A series of studies was undertaken to determine whether CO2 can be used as a humane as well as practical agent for euthanasia or anesthesia of rats. Human volunteers rated the degree of discomfort associated with breathing 50 to 100% CO2 mixed with oxygen. Increasing concentrations of CO2 were judged as progressively more noxious, from "highly unpleasant" for 50% CO2 to "painful" for 100% CO2. The practical aspects of anesthesia and euthanasia with 50 to 100% CO2 were studied, using male Sprague Dawley rats. Time to anesthesia and death were inversely related to CO2 concentration, as were the frequency and severity of adverse reactions, including seizures and hemorrhaging from the nose. The severity of edema and hemorrhage, which were observed on histologic examination of the lungs of all rats euthanized with CO2, were greatest in the animals exposed to the lowest concentrations. There were no significant effects of CO2 concentration on time to recumbency or recovery, and there were no significant effects of precharging versus not precharging the chamber on any of the parameters studied. It was concluded that, although CO2 can be used in a humane manner, the concentrations that are least likely to cause pain and distress are associated with the longest times to anesthesia and death, highest incidence of unwanted side effects, and most severe histologic changes in the lungs. Acceptably humane and reasonably practical euthanasia or anesthesia can be achieved using a nonprecharged chamber and a low gas flow rate so that conscious animals are never exposed to CO2 concentrations > 70%.