Ventilation rate (VR) is one of the two key elements for quantifying aerial emissions from animal production facilities. Direct, continuous measurement of building VR can be challenging and impractical under certain circumstances, e.g., naturally ventilated animal housing or a large number of ventilation fans in the building. This study examined the suitability of estimating VR of broiler houses with built-up litter (mixture of manure and bedding), when supplemental heating was not in use, through either carbon dioxide (CO 2 ) balance or the relationship of VR to CO 2 concentration difference between exhaust and inlet air. The reference VR was based on direct measurement by continuously monitoring operation of the in-situ calibrated exhaust fans. The comparative analysis of the direct method vs. each indirect method was conducted for a measurement integration time (MIT) of 10, 30, 60, or 120 min. The analyses revealed that MIT of 30 min or greater resulted in non-significant differences in VR between the indirect and direct methods. The broiler building VR (m 3 s -1 ) may be related to the exhaust-inlet CO 2 concentration difference ( ? CO 2 , ppm) as VR ( ± 3.0) = 4456 ( ± 41) ? CO 2 -0.786 ( ± 0.019) at 30 min MIT. The VR may also be determined by the CO2 balance method (including litter CO2 generation) with a correction factor of 0.97 at MIT of 30 to 120 min. If litter CO2 generation is omitted from the total building CO2 production, the actual VR may be estimated by applying a correction factor of 1.077 to the bird respiration CO2 balance VR.. Hence, the CO2 balance or concentration difference method offers a viable alternative or supplemental check for quantifying building VR under certain conditions where direct, continuous VR measurement is not feasible.