Summary Previously uncultured nitrite‐oxidizing bacteria affiliated to the genus Nitrospira have for the first time been successfully enriched from activated sludge from a municipal wastewater treatment plant. During the enrichment procedure, the abundance of the Nitrospira‐like bacteria increased to approximately 86% of the total bacterial population. This high degree of purification was achieved by a novel enrichment protocol, which exploits physiological features of Nitrospira‐like bacteria and includes the selective repression of coexisting Nitrobacter cells and heterotrophic contaminants by application of ampicillin in a final concentration of 50 µg ml−1. The enrichment process was monitored by electron microscopy, fluorescence in situ hybridization (FISH) with rRNA‐targeted probes and fatty acid profiling. Phylogenetic analysis of 16S rRNA gene sequences revealed that the enriched bacteria represent a novel Nitrospira species closely related to uncultured Nitrospira‐like bacteria previously found in wastewater treatment plants and nitrifying bioreactors. The enriched strain is provisionally classified as ‘Candidatus Nitrospira defluvii’.