Chemolithoautotrophic nitrite oxidizers of the genus Nitrospira are a monophyletic but diverse group of organisms, are widely distributed in many natural habitats, and play a key role in nitrogen elimination during biological wastewater treatment. Phylogenetic analyses of cloned 16S rRNA genes and fluorescence in situ hybridization with newly developed rRNA-targeted oligonucleotide probes revealed coexistence of uncultured members of sublineages I and II of the genus Nitrospira in biofilm and activated sludge samples taken from nitrifying wastewater treatment plants. Quantitative microscopic analyses of their spatial arrangement relative to ammonia oxidizers in the biofilm and activated sludge flocs showed that members of the Nitrospira sublineage I occurred significantly more often in immediate vicinity to ammonia oxidizers than would be expected from random community assembly while such a relationship was not observed for Nitrospira sublineage II. This spatial distribution suggested a niche differentiation of these coexisting Nitrospira populations with respect to their preferred concentrations of nitrite. This hypothesis was tested by mathematical modelling of nitrite consumption and resulting nitrite gradients in nitrifying biofilms and by quantifying the abundance of sublineage I and II Nitrospira in activated sludge during incubations with nitrite in different concentrations. Consistent with the observed localization patterns, a higher nitrite concentration selected for sublineage I but suppressed sublineage II Nitrospira.