Using prepared nitrifying sludge, anaerobic ammonia oxidization (anammox) sludge and two heterotrophic ammonia oxidization bacterial (AOB) species as inocula, this study elucidated the effect of oxygen conditions, assay media, and selective metabolic inhibitors on various microbial nitrogen (N)-transformation activities including aerobic chemolithotrophic ammonia and nitrite oxidization, aerobic heterotrophic ammonia oxidization, anammox, and aerobic and anoxic denitrification. The oxygen conditions and assay media effectively differentiated among almost all ammonia removal pathways except for separating aerobic chemolithotrophic ammonia oxidization from aerobic heterotrophic ammonia oxidization. A final allylthiourea concentration of 10 mg.L-1 was optimal for accurate determination of aerobic heterotrophic ammonia oxidization activity in the presence of aerobic chemolithotrophic AOB. Finally, this study developed a simple and reliable method to individually determine and compare the comprehensive N-transformation activity characteristics of several activated sludge samples from different origins, and to elucidate the major microbial N-transformation mechanisms for ammonia removal and N-2 production. (C) 2017 Elsevier Ltd. All rights reserved.