Direct measurement of building ventilation rate in livestock housing is a formidable task due to uncontrollable variations in fan and system performance. Estimating building ventilation rate based on a CO2 -balance offers a potentially viable alternative to direct measurement. Building ventilation rates obtained by direct measurement and by a CO2 balance were compared in this study for a commercial laying hen house with manure belts. The results indicate that ventilation rates estimated by the CO2 balance method were not significantly different (P>0.2) from those as determined by the direct measurement when the averaging or integration time interval was two hours or longer.= Careful application of the CO2 balance method will greatly improve the affordability and versatility of endeavors toward quantifying air emissions from confined animal housing.