Nitrous oxide (N2O), a strong greenhouse gas, can be produced by ammonium-oxidizing bacteria (AOB) as a by-product of ammonium oxidation and can potentially be formed in all types of nitrification processes. However, partial nitritation has been reported to cause significantly higher N2O emissions than complete nitrification. In the study presented here, the mechanisms and factors that drive N2O formation by AOB were investigated with respect to different operational strategies to achieve nitrite accumulation base on combined evaluation of oxygen uptake rate (OUR) and N2O formation rate. On the one hand, N2O formation during partial nitritation and nitrification in a continuously stirred tank reactor (CSTR) with continuous aerobic conditions was observed. On the other hand, the effect of intermittent aeration on N2O formation during nitrification was investigated. The presence of nitrite, the extend of sludge-specific ammonium loading, low oxygen concentration, and transition from aerobic to anoxic conditions significantly increased N2O formation in this reactor independently from each other, indicating that different formation pathways, supposedly via nitrite or hydroxylamine, were active.