Biological nitrogen removal (BNR) strategies can contribute to atmospheric nitrous oxide (N₂O). In the future, as BNR is implemented at more wastewater treatment plants (WWTPs) aro und the globe, the emission and release of these gases to the atmosphere is expected to increase. This study focused on the quantification of N₂O emissions rates and inventory at several WWTPs using a newly developed, U.S. Environmental Protection Agency (Washington, D.C.) (U.S.EPA)-reviewed protocol. Application of the protocol revealed nitrous oxide emissions from aerated zones therein, hitherto not considered in the current U.S. EPA approach to quantifying N₂O fluxes from WWTPs. A high degree of diurnal variability in N₂O fluxes was measured, which correlated well with diurnal total Kjeldahl nitrogen loadings. In combination, these results point to the diminishing utility of a single emission factor approach to estimate the N₂O emissions from WWTPs, which has been followed to date.