We present a novel 18O-15N-enrichment method for the distinction between nitrous oxide (N2O) from nitrification, nitrifier denitrification and denitrification based on a method with single- and double-15N-labelled ammonium nitrate. We added a new treatment with 18O-labelled water to quantify N2O from nitrifier denitrification. The theory behind this is that ammonia oxidisers use oxygen (O2) from soil air for the oxidation of ammonia (NH3), but use H2O for the oxidation of the resulting hydroxylamine (NH 2OH) to nitrite (NO2-). Thus, N2O from nitrification would therefore be expected to reflect the 18O signature of soil O2, whereas the 18O signature of N 2O from nitrifier denitrification would reflect that of both soil O2 and H2O. It was assumed that (a) there would be no preferential removal of 18O or 16O during nitrifier denitrification or denitrification, (b) the 18O signature of the applied 18O-labelled water would remain constant over the experimental period, and (c) any O exchange between H218O and NO3- would be negligible under the chosen experimental conditions. These assumptions were tested and validated for a silt loam soil at 50% water-filled pore space (WFPS) following application of 400 mg N kg -1 dry soil. We compared the results of our new method with those of a conventional inhibition method using 0.02% v/v acetylene (C2H 2) and 80% v/v O2 in helium. Both the 18O- 15N-enrichment and inhibitor methods identified nitrifier denitrification to be a major source of N2O, accounting for 44 and 40%, respectively, of N2O production over 24 h. However, compared to our 18O-15N-method, the inhibitor method overestimated the contribution from nitrification at the expense of denitrification, probably due to incomplete inhibition of nitrifier denitrification and denitrification by large concentrations of O2 and a negative effect of C 2H2 on denitrification. We consider our new 18O-15N-enrichment method to be more reliable than the use of inhibitors; it enables the distinction between more soil sources of N 2O than was previously possible and has provided the first direct evidence of the significance of nitrifier denitrification as a source of N 2O in fertilised arable soil.