In a model drinking water distribution system characterized by a low assimilable organic carbon content (<10 g/liter) and no disinfection, the bacterial community was identified by a phylogenetic analysis of rRNA genes amplified from directly extracted DNA and colonies formed on R2A plates. Biofilms of defined periods of age (14 days to 3 years) and bulk water samples were investigated. Culturable bacteria were associated with Proteobacteria and Bacteriodetes, whereas independently of cultivation, bacteria from 12 phyla were detected in this system. These included Acidobacteria, Nitrospirae, Planctomycetes, and Verrucomicrobia, some of which have never been identified in drinking water previously. A cluster analysis of the population profiles from the individual samples divided biofilms and bulk water samples into separate clusters (P ؍ 0.027). Bacteria associated with Nitrospira moscoviensis were found in all samples and encompassed 39% of the sequenced clones in the bulk water and 25% of the biofilm community. The close association with Nitrospira suggested that a large part of the population had an autotrophic metabolism using nitrite as an electron donor. To test this hypothesis, nitrite was added to biofilm and bulk water samples, and the utilization was monitored during 15 days. A first-order decrease in nitrite concentration was observed for all samples with a rate corresponding to 0.5 ؋ 10 5 to 2 ؋ 10 5 nitrifying cells/ml in the bulk water and 3 ؋ 10 5 cells/cm 2 on the pipe surface. The finding of an abundant nitrite-oxidizing microbial population suggests that nitrite is an important substrate in this system, potentially as a result of the low assimilable organic carbon concentration. This finding implies that microbial communities in water distribution systems may control against elevated nitrite concentrations but also contain large indigenous populations that are capable of assisting the depletion of disinfection agents like chloramines.Drinking water is an important resource all around the globe. Nevertheless, little research has focused on identifying the bacteria in water distribution systems or how treatment techniques alter the composition. In contrast, the bacteria present in wastewater treatment plants are very well characterized by a suite of advanced molecular techniques (40). The monitoring and identification of bacteria present in drinking water have primarily been achieved using a plating and isolation strategy. The majority of these culturable bacteria described in drinking water networks are included in the phylum Proteobacteria and to a lesser extent in the phyla Actinobacteria, Firmicutes, and Bacteriodetes (18,19,29,30,32). Some of the commonly detected genera include Pseudomonas, Caulobacter, Aeromonas, Acinetobacter, and Bacillus. As a result, a common perception is that Pseudomonas is the most abundant bacterial organism in supply systems, regardless of the water source.It is well known that the plate count technique significantly underestimates the total number of cells prese...