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CO2, a byproduct of respiration, is toxic at high concentrations so regulation of CO2 within the honey bee hive is an important colony function. In this study, we measured hive CO2 concentrations at 1-s intervals while ventilation characteristics of the hive were changed every few days, and we analyzed the data for effects of increased ventilation on colony behavior and thermoregulation. Average CO2 concentrations were significantly higher, by > 200 ppm, when hives had screened bottom boards (higher ventilation) compared to hives with solid bottom boards (lower ventilation) at the same time. Daily CO2 concentration amplitudes, hourly temperature, daily temperature amplitudes, nor hourly hive weight changes were not significantly affected by the changes in hive ventilation. In a second experiment, we found average CO2 concentrations at the top center of the upper hive box, on top of the frames, were significantly lower than concentrations at the center of a solid bottom board underneath frames, which was expected due to the higher density of CO2 relative to air. Bee colonies have been reported to cycle air, with shorter periods of 20 to 150 s and longer periods of 42–80 min, but a periodogram analysis of the CO2 concentration data found no evidence of important CO2 cycle periods other than a strong 24-h period. Bee colonies maintained strong daily cycles of CO2 concentration, with average maximum concentrations > 11,000 ppm, even in conditions of increased ventilation, indicating that managing CO2 concentration is a complex colony behavior. [ABSTRACT FROM AUTHOR]