The inhibitory effects of nitrite (NO2-)/free nitrous acid (HNO2-FNA) on the metabolism of Nitrobacter were investigated using a method allowing the decoupling of the growth and energy generation processes. A lab-scale sequencing batch reactor was operated forthe enrichment of a Nitrobacter culture. Fluorescent in situ hybridization (FISH) analysis showed that 73% of the bacterial population was Nitrobacter. Batch tests were carried out to assess the oxygen and nitrite consumption rates of the enriched culture at low and high nitrite levels, in the presence or absence of inorganic carbon. It was observed that in the absence of CO2, the Nitrobacter culture was able to oxidize nitrite at a rate that is 76% of that in the presence of CO2, with an oxygen consumption rate that is 85% of that measured in the presence of CO2. This enabled the impacts of nitrite/FNA on the catabolic and anabolic processes of Nitrobacter to be assessed separately. FNA rather than nitrite was likely the actual inhibitor to the Nitrobacter metabolism. It was revealed that FNA of up to 0.05 mg HNO2-N x L(-1) (3.4 microM), which was the highest FNA concentration used in this study, did not have any inhibitory effect on the catabolic processes of Nitrobacter. However, FNA initiated its inhibition to the anabolic processes of Nitrobacterat approximately 0.011 mg HNO2-N x L(-1) (0.8 microM), and completely stopped biomass synthesis at a concentration of approximately 0.023 mg HNO2-N x L(-1) (1.6 microM). The inhibitory effect could be described by an empirical inhibitory model proposed in this paper, but the underlying mechanisms remain to be revealed.