Microautoradiography combined with fluorescence in situ hybridization (MAR-FISH) was used to screen for potential polyphosphate-accumulating organisms (PAO) in a full-scale enhanced biological phosphorus removal (EBPR) plant. The results showed that, in addition to uncultured Rhodocyclus-related PAO, two morphotypes hybridizing with gene probes for the gram-positive Actinobacteria were also actively involved in uptake of orthophosphate (P i ). Clone library analysis and further investigations by MAR-FISH using two new oligonucleotide probes revealed that both morphotypes, cocci in clusters of tetrads and short rods in clumps, were relatively closely related to the genus Tetrasphaera within the family Intrasporangiaceae of the Actinobacteria (93 to 98% similarity in their 16S rRNA genes). FISH analysis of the community biomass in the treatment plant investigated showed that the short rods (targeted by probe Actino-658) were the most abundant (12% of all Bacteria hybridizing with general bacterial probes), while the cocci in tetrads (targeted by probe Actino-221) made up 7%. Both morphotypes took up P i aerobically only if, in a previous anaerobic phase, they had taken up organic matter from wastewater or a mixture of amino acids. They could not take up short-chain fatty acids (e.g., acetate), glucose, or ethanol under anaerobic or aerobic conditions. The storage compound produced during the anaerobic period was not polyhydroxyalkanoates, as for Rhodocyclus-related PAO, and its identity is still unknown. Growth and uptake of P i took place in the presence of oxygen and nitrate but not nitrite, indicating a lack of denitrifying ability. A survey of the occurrence of these actinobacterial PAO in 10 full-scale EBPR plants revealed that both morphotypes were widely present, and in several plants more abundant than the Rhodocyclus-related PAO, thus playing a very important role in the EBPR process.In the wastewater treatment industry, enhanced biological phosphorus removal (EBPR) has been widely used to remove orthophosphate (P i ) from wastewater to protect the receiving water bodies against eutrophication. In EBPR processes, the principle is to enrich microorganisms that can accumulate excessive amounts of intracellular polyphosphate [poly(P)] by using sequential anaerobic-aerobic and/or anaerobic-denitrifying conditions (37). The understanding of the biochemical pathways of these poly(P)-accumulating organisms (PAO) is based mainly on analysis of chemical transformations in enriched laboratory-scale EBPR systems, because no pure cultures are available (25, 34, 37). The generally accepted hypothesis (25, 34) proposes that PAO take up organic matter (usually assumed to be acetate) during the anaerobic period by using poly(P) as an energy source and sequester the acetate taken up into polyhydroxyalkanoates (PHA). The reducing power to form PHA is provided by hydrolysis of an intracellular glycogen pool through the glycolytic pathway. The PHA are used as energy and carbon sources in the following aerobic or denitrifying...