Nitric oxide (NO) and nitrous oxide (N2O) emissions from nitrifying ecosystems are a serious threat to the environment. The factors influencing the emission and the responsible microorganisms and pathways were studied using a laboratory-scale nitrifying reactor system. The nitrifying culture was established at growth rates relevant to wastewater treatment plants (WWTPs). During stable ammonia oxidation, 0.03% of ammonium was emitted as NO and 2.8% was emitted as N2O. Although mixed cultures were used, clear responses in emission of ammonia oxidizing bacteria (AOB) could be detected and it was concluded that the denitrification pathway of AOB was the main source of the emissions. Emissions of nitrogen oxides in the system were strongly influenced by oxygen, nitrite, and ammonium concentrations. Steady state emission levels greatly underestimate the total emission, because changes in oxygen, nitrite, and ammonium concentrations induced a dramatic rise in NO and N2O emission. The data presented can be used as an indication for NO and N2O emission by AOB in plug-flow activated sludge systems, which is highly relevant because of the atmospheric impact and potential health risk of these compounds.