The long-term contribution of nitrification to nitrous oxide (N₂O) emissions from terrestrial ecosystems is poorly known and thus poorly constrained in biogeochemical models. Here, using Bayesian inference to couple 25 years of in situ N₂O flux measurements with site-specific Michaelis-Menten kinetics of nitrification-derived N₂O, we test the relative importance of nitrification-derived N₂O across six cropped and unmanaged ecosystems along a management intensity gradient in the U.S. Midwest. We found that the maximum potential contribution from nitrification to in situ N₂O fluxes was 13-17% in a conventionally fertilized annual cropping system, 27-42% in a low-input cover-cropped annual cropping system, and 52-63% in perennial systems including a late successional deciduous forest. Actual values are likely to be less than 10% of these values because of low N₂O yields in cultured nitrifiers (typically 0.04 to 8% of NH₃ oxidized) and competing sinks for available NH₄⁺ in situ. Most nitrification-derived N₂O was produced by ammonia oxidizing bacteria (AOB) rather than archaea (AOA), who appeared responsible for no more than 30% of nitrification-derived N₂O production in all but one ecosystem. Although the proportion of nitrification-derived N₂O production was lowest in annual cropping systems, these ecosystems nevertheless produced more nitrification-derived N₂O (higher V_max) than perennial and successional ecosystems. We conclude that nitrification is minor relative to other sources of N₂O in all ecosystems examined.

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