We present a novel O-18-N-15-enrichment method for the distinction between nitrous oxide (N2O) from nitrification, nitrifier denitrification and denitrification based on a method with single- and double-N-15-labelled ammonium nitrate. We added a new treatment with O-18-labelled water to quantify N2O from nitrifier denitrification. The theory behind this is that ammonia oxidisers use oxygen (O-2) from soil air for the oxidation of ammonia (NH3) but use H2O for the oxidation of the resulting hydroxylamine (NH2OH) to nitrite (NO2-). Thus, N2O from nitrification would therefore be expected to reflect the O-18 signature of soil O-2, whereas the O-18 signature of N2O from nitrifier denitrification would reflect that of both soil O-2 and H2O. It was assumed that (a) there would be no preferential removal of O-18 or O-16 during nitrifier denitrification or denitrification, (b) the O-18 signature of the applied O-18-labelled water would remain constant over the experimental period, and (c) any O exchange between (H2O)-O-18 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 (C2H2) and 80% v/v O-2 in helium. Both the O-18-N-15-enrichment and inhibitor methods identified nitrifter denitrification to be a major source of N2O, accounting for 44 and 40%, respectively, of N2O production over 24 h. However, compared to our O-18-N-15-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 02 and a negative effect of C2H2 on denitrification. We consider our new O-18-N-15-enrichment method to be more reliable than the use of inhibitors; it enables the distinction between more soil sources of N2O than was previously possible and has provided the first direct evidence of the significance of nitrifier denitrification as a source of N2O in fertilised arable soil.