External carbon sources can enhance denitrification rates and thus improve nitrogen removal in wastewater treatment plants. The effects of adding methanol and ethanol on the genetic and metabolic diversity of denitrifying communities in activated sludge were compared using a pilot-scale plant with two parallel lines. A full-scale plant receiving the same municipal wastewater, but without external carbon source addition, was the reference. Metabolic profiles obtained from potential denitrification rates with 10 electron donors showed that the denitrifying communities altered their preferences for certain compounds after supplementation with methanol or ethanol and that methanol had the greater impact. Clone libraries of nirK and nirS genes, encoding the two different nitrite reductases in denitrifiers, revealed that methanol also increased the diversity of denitrifiers of the nirS type, which indicates that denitrifiers favored by methanol were on the rise in the community. This suggests that there might be a niche differentiation between nirS and nirK genotypes during activated sludge processes. The composition of nirS genotypes also varied greatly among all samples, whereas the nirK communities were more stable. The latter was confirmed by denaturing gradient gel electrophoresis of nirK communities on all sampling occasions. Our results support earlier hypotheses that the compositions of denitrifier communities change during predenitrification processes when external carbon sources are added, although no severe effect could be observed from an operational point of view.Considerable efforts have been made to improve the technology for efficient and economic removal of nitrogen by denitrification in municipal activated sludge. Denitrification is an anaerobic microbial respiration process with a stepwise reduction of nitrate or nitrite via nitric oxide to nitrous oxide or dinitrogen. In the ideal activated sludge process, bacteria denitrify by using carbon compounds in the influent wastewater as electron donors. As a means to improve control strategies for nitrogen removal, external carbon compounds can be added to enhance denitrification rates (10,14,18,19,34,35). Higher rates allow reductions in the hydraulic retention time in the anoxic zones, making it possible to minimize basin volumes (23). For this purpose, methanol and ethanol are most commonly used in practice, although acetate has been reported to give the highest rates in most cases (12,24,(28)(29)(30).Although much is known about process performance after the addition of external carbon sources, little effort has been made to explain the specific effects on the denitrifying communities in activated sludge. The selective pressure from the carbon compound added might affect the composition of the denitrifying community, which would influence metabolic diversity and functional properties of the denitrifiers. For example, it was shown that acetate as the major carbon source in a laboratory-scale reactor selected for denitrifiers with nonflocculating proper...