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Nitrifier denitrification (i.e. nitrite reduction by ammonia oxidizers) is one of the biochemical pathways of nitrous oxide (N2O) production. It is increasingly suggested that this pathway may contribute substantially to N2O production in soil, the major source of this greenhouse gas. However, although monoculture studies recognize its potential, methodological drawbacks prohibit conclusive proof that nitrifier denitrification occurs in actual soils. Here we suggest and apply a new isotopic approach to identify its presence in soil. In incubation experiments with 12 soils, N2O production was studied using oxygen (O) and nitrogen (N) isotope tracing, accounting for O exchange. Microbial biomass C and N and phospholipid fatty acid (PLFA) patterns were analysed to explain potential differences in N2O production pathways. We found that in at least five of the soils nitrifier denitrification must have contributed to N2O production. Moreover, it may even have been responsible for all NH4+-derived N2O in most soils. In contrast, N2O as a by-product of ammonia oxidation contributed very little to total production. Microbial biomass C and N and PLFA-distinguished microbial community composition were not indicative of differences in N2O production pathways. Overall, we show that combined O and N isotope tracing may still provide a powerful tool to understand N2O production pathways, provided that O exchange is accounted for. We conclude that nitrifier denitrification can indeed occur in soils, and may in fact be responsible for the greater proportion of total nitrifier-induced N2O production.