Carbon Dioxide Gas is Used as an Insect Anesthetic in Many Laboratories, Despite Recent Studies Which Have Shown that CO2 Can Alter Behavior and Fitness. We Examine the Effects of CO2 and Anoxia (N2) on Cold Tolerance, Measuring the Rapid Cold-Hardening (RCH) Response and Chill Coma Recovery in Drosophila Melanogaster. Short Exposures to CO2 or N2 Do Not Significantly Affect RCH, But 60 Min of Exposure Negates RCH. Exposure to CO2 Anesthesia Increases Chill Coma Recovery Time, But This Effect Disappears If the Flies Are Given 90 Min Recovery in Air Before Chill Coma Induction. Flies Treated with N2 Show a Similar Pattern, But Require Significantly Longer Chill Coma Recovery Times Even after 90 Min of Recovery from Anoxia. Our Results Suggest that CO2 Anesthesia is an Acceptable Way to Manipulate Flies Before Cold Tolerance Experiments (When using RCH or Chill Coma Recovery as a Measure), Provided Exposure Duration is Minimized and Recovery is Permitted Before Chill Coma Induction. However, We Recommend that Exposure to N2 Not Be Used as a Method of Anesthesia for Chill Coma Studies. © 2006 Elsevier Ltd. All Rights Reserved.