Cu inhibition of gene transcription in ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) were rarely studied simultaneously in activated sludge. In this study, the transcription of amoA (for AOB) and nxrB (for NOB), nitrification efficiencies, AOB and NOB respiratory rates, and Cu distribution were simultaneously investigated. Modeling the relationships among the aforementioned parameters revealed that in complex activated sludge systems, nitrification efficiency was an insensitive parameter for showing Cu inhibition. Respiration activities and gene transcription were sensitive to Cu and positively correlated with each other. The transcription of amoA and nxrB genes indicated that the Cu had different inhibitory effects on AOB and NOB. AOB were more susceptible to Cu toxicity than NOB. Moreover, the degree of Cu inhibition on ammonia oxidation was greater than on nitrite oxidation. The analysis and related modeling results indicate that the inhibitory actions of Cu on nitrifying bacteria could mainly be attributed to intracellular Cu. The findings from this study provide insight into the mechanism of Cu inhibition on nitrification in complex activated sludge systems. (C) 2015 Elsevier B.V. All rights reserved.