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Natural oestrogens, which are degraded but not completely removed in wastewater treatment plants, are suspected of causing the endocrine disruption of aquatic organisms in the receiving water body. While several bacterial isolates were reported to be oestrogen-degrading bacteria, our previous study implied that only the unidentified rod-shaped Betaproteobacteria in chains were responsible for estrone (E1) degradation by activated sludge especially at the sub-milligram per litre level. The Betaproteobacteria were suspected to be related to genera Sphaerotilus and Leptothrix according to morphological observations. Probe Spha823 was newly developed to target 16S rRNA gene clones obtained from activated sludge and closely related to the above genera. [3H]E1-incubated sludge samples showed that most of the 3H-labelled cells hybridized with probe Spha823 by microautoradiography (MAR) fluorescent in situ hybridization. Spha823-defined cells were present in all three activated sludge samples tested, where they accounted for up to 3% of the total microbial biomass. Spha823-defined cells comprised 59·5–80·1% of the total MAR-positive cells, which suggested that the Sphaerotilus–Leptothrix-related bacteria were the most abundant micro-organisms involved in E1 degradation (at 200 μg l−1) in the activated sludge samples.Significance and Impact of the Study: Estrone (E1) is one of the natural estrogens, which can be degraded but is not always completely removed in wastewater treatment plants. E1 is suspected of causing the endocrine disruption of aquatic organisms in the receiving water body. We identified dominant E1-incorporating bacteria, which should include E1-degrading bacteria, in activated sludge treating domestic wastewater. Sphaerotilus–Leptothrix-related bacteria, which had never been reported in the previous attempts based on culture-dependent approach, occupied 60–80% of the E1-incorporating bacteria. This study demonstrates the identification of functionally active bacteria to degrade micro-pollutants at sub-milligram per litre level.