The purpose of this study was to investigate the formation of nitrous oxide (N2O) in nitritation and nitrification under stable, comparable and not limiting conditions typical for treatment of high-strength wastewater. A laboratory-scale aerated chemostat was operated with reject water at different sludge retention times, achieving suppression of nitrate formation by wash-out of nitrite-oxidizing bacteria for nitritation. The N2O formation factor during stable nitritation was higher (2.90% N2O-N /NH4−-Nox) than during nitrification (0.74%). The positive correlation of N2O formation rates and ammonium oxidation rates was linear and thus did not contribute to changes of the N2O formation factor. The dominant factor for N2O formation during stable operation was high nitrite concentration, which was positively correlated with N2O formation rates. The highest formation factors were observed during a transition phase from nitrification to nitritation with unstable process conditions (4.81%) and during a short-term experiment with increased pH of 7 (10.28%). The results indicate that even with operational conditions that are regarded favourable for the process of nitritation N2O formation can be limited but not avoided.