A laboratory scale reactor operated as a single sludge, denitrification-nitrification bioreactor (DNB), was fed a synthetic wastewater. The effect of the C/N ratio of the influent on the structure of beta-proteobacterial autotrophic ammonia-oxidizing bacterial (AOB) communities was determined by DGGE analysis of 16S rRNA gene fragments amplified using a range of AOB-selective primers. Fluorescence in situ hybridisation (FISH) was used to determine quantitative changes in the AOB communities. When operated at a C/N ratio of 2 the DNB was effective in nitrogen removal and nitrification was measured at approximately 1.0 mg NH4+-N/g dry wt/h. Altering the C/N ratio to 5 resulted in a 50% reduction in nitrification rates. Nitrification was restored to its original level when the C/N ratio was returned to 2. AOB were detected by DGGE analysis of samples from the DNB under all operating conditions but the changes in C/N ratio and nitrification rates were accompanied by changes in the community structure of the AOB. However, quantitative FISH analysis indicated that beta-proteobacterial AOB were only present in high numbers (ca. 10(8) cells/ml) under the original operating conditions with a C/N ratio of 2. Beta-proteobacterial AOB could not be detected by FISH when the C/N ratio was 5. When nitrification activity was restored by returning the C/N ratio to 2, beta-proteobacterial AOB were still not detected and it is likely that either beta-proteobacterial AOB were not responsible for ammonia oxidation or that beta-proteobacterial AOB that did not contain the target sites for the range of 4 AOB selective probes used, were present in the reactor.