The in situ ecophysiology of alphaproteobacterial filamentous Cluster III Defluviicoccus present in enhanced biological phosphorus removal (EBPR)-activated sludge systems was evaluated using FISH-MAR and histochemical staining methods. These organisms, sharing the Nostocoida limicola morphotype, are known to be responsible for serious episodes of activated sludge bulking. The data presented here also demonstrate an ability to assimilate short-chain fatty acids and synthesize poly-β-hydroxyalkanoates (PHA) anaerobically, and then utilize this stored PHA under aerobic conditions, but with no corresponding synthesis of polyphosphate. These features are consistent with an in situ phenotype of glycogen-accumulating organisms (GAO), populations thought to lower the efficiency of EBPR systems by outcompeting polyphosphate-accumulating organisms (PAO) for substrates in their anaerobic feed phase. Survey data indicate that these GAO are as commonly seen as the known PAO in full-scale EBPR-activated sludge systems, which suggest that they might play important roles there, and therefore should not be viewed just as laboratory curiosities.