Despite differences in their morphologies, comparative analyses of 16S rRNA gene sequences revealed high levels of similarity (>94 %) between strains of the filamentous bacterium ‘CandidatusNostocoida limicola’ and the cocciTetrasphaera australiensisandTetrasphaera japonicaand the rodTetrasphaera elongata, all isolated from activated sludge. These sequence data and their chemotaxonomic characters, including cell wall, menaquinone and lipid compositions and fingerprints of their 16S–23S rRNA intergenic regions, support the proposition that these isolates should be combined into a single genus containing six species, in the familyIntrasporangiaceaein theActinobacteria. This suggestion receives additional support from DNA–DNA hybridization data and when partial sequences of therpoC1gene are compared between these strains. Even though few phenotypic characterization data were obtained for these slowly growing isolates, it is proposed, on the basis of the extensive chemotaxonomic and molecular evidence presented here, that ‘CandidatusN. limicola’ strains Ben 17, Ben 18, Ben 67, Ben 68 and Ben 74 all be placed into the speciesTetrasphaera jenkinsiisp. nov. (type strain Ben 74T=DSM 17519T=NCIMB 14128T), ‘CandidatusN. limicola’ strain Ben 70 intoTetrasphaera vanveeniisp. nov. (type strain Ben 70T=DSM 17518T=NCIMB 14127T) and ‘CandidatusN. limicola’ strains Ver 1 and Ver 2 intoTetrasphaera veronensissp. nov. (type strain Ver 1T=DSM 17520T=NCIMB 14129T).