Nitrous oxide (N2O) emissions from a nitrifying biofilm reactor were investigated with N2O isotopocules. The nitrogen isotopomer site preference of N2O (N-15-SP) indicated the contribution of producing and consuming pathways in response to changes in oxygenation level (from 0% to 21% O-2 in the gas mix), temperature (from 13.5 to 22.3 degrees C) and ammonium concentrations (from 6.2 to 62.1 mg N L-1). Nitrite reduction, either nitrifier denitrification or heterotrophic denitrification, was the main N2O-producing pathway under the tested conditions. Difference between oxidative and reductive rates of nitrite consumption was discussed in relation to NO2- concentrations and N2O emissions. Hence, nitrite oxidation rates seem to decrease as compared to ammonium oxidation rates at temperatures above 20 degrees C and under oxygen-depleted atmosphere, increasing N2O production by the nitrite reduction pathway. Below 20 degrees C, a difference in temperature sensitivity between hydroxylamine and ammonium oxidation rates is most likely responsible for an increase in N2O production via the hydroxylamine oxidation pathway (nitrification). A negative correlation between the reaction kinetics and the apparent isotope fractionation was additionally shown from the variations of delta N-15 and delta O-18 values of N2O produced from ammonium. The approach and results obtained here, for a nitrifying biofilm reactor under variable environmental conditions, should allow for application and extrapolation of N2O emissions from other systems such as lakes, soils and sediments.