N 2 O is an important greenhouse gas and the primary stratospheric ozone depleting substance. Its deleterious effects on the environment have prompted appeals to regulate emissions from agriculture, which represents the primary anthropogenic source in the global N 2 O budget. Successful implementation of mitigation strategies requires robust bottom-up inventories that are based on emission factors (EFs), simulation models, or a combination of the two. Top-down emission estimates, based on tall-tower and aircraft observations, indicate that bottom-up inventories severely underestimate regional and continental scale N 2 O emissions, implying that EFs may be biased low. Here, we measured N 2 O emissions from streams within the US Corn Belt using a chamber-based approach and analyzed the data as a function of Strahler stream order (S). N 2 O fluxes from headwater streams often exceeded 29 nmol N 2 O-N m −2 ·s −1 and decreased exponentially as a function of S. This relation was used to scale up riverine emissions and to assess the differences between bottom-up and top-down emission inventories at the local to regional scale. We found that the Intergovernmental Panel on Climate Change (IPCC) indirect EF for rivers (EF 5r ) is underestimated up to ninefold in southern Minnesota, which translates to a total tier 1 agricultural underestimation of N 2 O emissions by 40%. We show that accounting for zero-order streams as potential N 2 O hotspots can more than double the agricultural budget. Applying the same analysis to the US Corn Belt demonstrates that the IPCC EF 5r underestimation explains the large differences observed between top-down and bottom-up emission estimates.aquatic nitrous oxide fluxes | IPCC emission factors | river emission hotspots | regional emission upscaling N 2 O is projected to remain the dominant stratospheric ozonedepleting substance of the 21st century (1) and is a powerful greenhouse gas (GHG) that currently accounts for about 6% of the net radiative forcing associated with long-lived anthropogenic GHGs (2). The detrimental environmental impacts of N 2 O have stimulated appeals to regulate emissions from agricultural lands (1, 3), which account for nearly 80% of the global anthropogenic N 2 O budget (4, 5). The successful regulation and mitigation of N 2 O emissions requires a sound understanding of the direct and indirect emission processes and reduced uncertainty regarding the emission factors (EFs) (6).The Intergovernmental Panel on Climate Change (IPCC) tier 1 approach uses EFs to provide first-order approximations of annual N 2 O emissions based on mechanistic and empirical information that have been constrained by field studies. These EFs are widely used in bottom-up inventories such as the Emission Database for Global Atmospheric Research (EDGAR) (7) and the Global Emissions Initiative (GEIA) (8). These inventories are essential tools for tracking country specific emission trends, assessing thresholds for international treaties, and evaluating the impacts of mitigation policies. Recent i...