While recognised as the important population responsible for enhanced biological phosphorus removal (EBPR), detailed knowledge on the physiology of Rhodocyclus-related polyphosphate accumulating organisms (PAO) has yet to be grasped. The objective of this study was to examine the in situ substrate uptake patterns of Rhodocyclus-related PAO present in full-scale EBPR plants by the combined technique of microautoradiography-fluorescent in situ hybridization (MAR-FISH). The presence of these PAO in the four investigated plants was confirmed by FISH and they constituted 17%, 9%, 8%, and 7% of the sludge community. By using MAR-FISH technique, Rhodocyclus-related PAO in all the plants demonstrated similar anaerobic substrate uptake patterns. They were capable of assimilating acetate, aspartate and glutamate under anaerobic condition but they showed negative uptake with palmitate. A significant fraction of the MAR-positive cells assimilated acetate, aspartate or glutamate was found to be Rhodocyclus-related PAO. Dual staining with DAPI and FISH showed that these PAO also accumulated polyphosphate aerobically with aspartate and glutamate as carbon source. The ability of assimilating amino acids besides acetate strongly indicates the versatile physiology of Rhodocyclus-related PAO, which could benefit them to achieve predominance in EBPR activated sludge.