Direct measurement of building ventilation rate in livestock housing is a formidable task due to uncontrollable variations in fan and system performance that are caused by factors such as building static pressure, fan belt slippage, and dust accumulation on shutters and blades. Estimating building ventilation rate by an indirect method based on a CO2 balance offers a potentially viable alternative to direct measurement. The validity of the CO2 balance method depends on the validity of relationship between CO2 production inside the building and metabolic rate of the animals and the knowledge of CO2 generation by the housing environment. Metabolic rates of modern laying hens have recently been quantified in intensive large-scale laboratory measurements. However, performance of the indirect method remains to be evaluated under field conditions. This article compares building ventilation rates obtained by direct measurement and by a CO2 balance. The test was conducted at a commercial laying hen house that used manure belts with daily manure removal. The results indicate that ventilation rates estimated by the indirect method were not significantly different (P > 0.2) from those as determined by the direct measurement when the averaging or integration time interval was 2 h or longer. Careful application of the indirect method could greatly improve the affordability and versatility of endeavors toward quantifying air emissions from confined animal housing.