N₂O δ¹⁵N and δ¹⁸O values changed non-linearly during in vitro N₂O production by bacterial denitrification of NO₃⁻, an argument against their use in N₂O apportionment. We present a novel approach for describing non-linear isotopic behaviour, which may be applied to other multi-step reactions. We also show that the intramolecular distribution of ¹⁵N in N₂O, used to apportion N₂O emissions to denitrification and nitrification, is unaffected by electron donor source and electron donor concentration.