Nitric oxide production was measured during nitrification in a laboratory-scale bioreactor, operated at conditions relevant to municipal nitrifying wastewater treatment plants. This study aims to determine which type of microorganism and which metabolic pathway is responsible for nitric oxide emission during nitrification. Simulation studies were used to identify which pathway is the main source of nitric oxide emission, based on the following three hypothetical pathways for nitric oxide emission: (a) nitrification, (b) denitrification by ammonia-oxidizing bacteria with ammonium as electron donor, and (c) heterotrophic denitrification. The results of the study suggest that, in a nitrifying reactor treating wastewater containing solely ammonium and nutrients, denitrification by ammonia-oxidizing bacteria is the main nitric-oxide-producing pathway. During the experiments, 0.025% of the treated ammonium is emitted as nitric oxide, independent of the aeration rate imposed. Nitrite presence and oxygen limitation were found to increase the nitric oxide emission.