ABSTRACT Chemolithoautotrophic nitrifying bacteria release soluble organic compounds, which can be substrates for heterotrophic microorganisms. The identities of these heterotrophs and the specificities of their interactions with nitrifiers are largely unknown. In this study, we incubated nitrifying activated sludge with ¹³ C-labeled bicarbonate and used stable isotope probing of 16S rRNA to monitor the flow of carbon from uncultured nitrifiers to heterotrophs. To facilitate the identification of heterotrophs, the abundant 16S rRNA molecules from nitrifiers were depleted by catalytic oligonucleotides containing locked nucleic acids (LNAzymes), which specifically cut the 16S rRNA of defined target organisms. Among the ¹³ C-labeled heterotrophs were organisms remotely related to Micavibrio , a microbial predator of Gram-negative bacteria. Fluorescence in situ hybridization revealed a close spatial association of these organisms with microcolonies of nitrite-oxidizing sublineage I Nitrospira in sludge flocs. The high specificity of this interaction was confirmed by confocal microscopy and a novel image analysis method to quantify the localization patterns of biofilm microorganisms in three-dimensional (3-D) space. Other isotope-labeled bacteria, which were affiliated with Thermomonas , colocalized less frequently with nitrifiers and thus were commensals or saprophytes rather than specific symbionts or predators. These results suggest that Nitrospira spp. are subject to bacterial predation, which may influence the abundance and diversity of these nitrite oxidizers and the stability of nitrification in engineered and natural ecosystems. In silico screening of published next-generation sequencing data sets revealed a broad environmental distribution of the uncultured Micavibrio -like lineage.