The first step of nitrification is catalysed by both ammonia-oxidizing bacteria (AOB) and archaea (AOA), but physicochemical controls on the relative abundance and function of these two groups are not yet fully understood, especially in freshwater environments. This study investigated ammonia-oxidizing populations in nitrifying rotating biological contactors (RBCs) from a municipal wastewater treatment plant. Individual RBC stages are arranged in series, with nitrification at each stage creating an ammonia gradient along the flowpath. This RBC system provides a valuable experimental system for testing the hypothesis that ammonia concentration determines the relative abundance of AOA and AOB. The results demonstrate that AOA increased as ammonium decreased across the RBC flowpath, as indicated by qPCR for thaumarchaeal amoA and 16S rRNA genes, and core lipid (CL) and intact polar lipid (IPL) crenarchaeol abundances. Overall, there was a negative logarithmic relationship (R2 = 0.51) between ammonium concentration and the relative abundance of AOA amoA genes. A single AOA population was detected in the RBC biofilms; this phylotype shared low amoA and 16S rRNA gene homology with existing AOA cultures and enrichments. These results provide evidence that ammonia availability influences the relative abundances of AOA and AOB, and that AOA are abundant in some municipal wastewater treatment systems.