An on-line NAD(P)H fluorometer has been used to investigate the fluorescence profile of a simulated anaerobic/anoxic/oxic biological nutrient removal (BNR) process and its dependency on operating parameters, such as the concentrations of organic substrate (acetate) and activated sludge. As expected from the different rates of NAD(P)H oxidation at different stages, the order of fluorescence levels was found to be anaerobic > anoxic > oxic. The effects of the acetate and sludge concentrations were observed most clearly at the anoxic stage : an increase in either concentration led to faster recovery of fluorescence from the drop caused by NO3- addition. A higher acetate concentration also resulted in a more significant, continuous rise in fluorescence, corresponding well with the enhanced PO43- release, at the anaerobic stage and a slower initial fluorescence decrease at the oxic stage, indicating the dependency of the decrease on substrate availability. The ratio of the two fluorescence drops caused by NO3- addition and aeration, Delta(1)/Delta(2), obtained with the plant’s sludge was found to be 0.7 +/- 0.1, which is similar to those of pure nitrate-respiring cultures (e.g., Pseudomonas aeruginosa and Escherichia coli). The dominant majority of non-nitrifiers in the sludge therefore are capable of nitrate respiration, and the nitrifiers either contribute little to the overall fluorescence or perform nitrate respiration as the rest of the sludge population.