Large, homogenous clusters of coccobacilli were found to be abundant in the biomasses from a conventional plant at Rosebud, Victoria, Australia. The identity and the in situ physiology of these dominant microorganisms were investigated in this study. These large clustered cells were revealed to be neither Gram positive nor Gram negative bacteria and contain polyP granules. Cells with similar features were also observed in some enhanced biological phosphate removal (EBPR) systems and reported as yeast spores and Rhodocyclus-related polyphosphate accumulating organisms (PAOs). In this study, fluorescent in situ hybridization (FISH) probing showed these cells were prokaryotic and members of the beta-Proteobacteria. However, these large clustered cells did not respond to the PAO mix FISH probes. The in situ physiology of these large cells was studied with FISH in combination with microautoradiography (MAR) in order to understand their substrate assimilation abilities under different conditions as well as their phosphate uptake ability. These cells were able to take up acetate, glutamate and aspartate, but not glucose under both aerobic and anaerobic conditions. Nile Blue A staining in combination with MAR showed that cells incubated under anaerobic conditions contained polyhydroxyalkanoates (PHA) granules. In addition, MAR showed aerobic 33Pi assimilation with all these substrates, consistent with them supporting an EBPR capacity in these large cells. As well as raising doubts about a role for yeasts in EBPR, this study suggests that much still needs to be learned about the identity and level of biodiversity of the PAO in EBPR systems, and emphasizes the benefits of using techniques like FISH/MAR and PHA staining/MAR to resolve the in situ physiology of the populations of interest there.