This study analysed the enhanced biological phosphorus removal (EBPR) microbial community and metabolic performance of five full-scale EBPR systems by using fluorescence in situ hybridisation combined with off-line batch tests fed with acetate under anaerobic-aerobic conditions. The phosphorus accumulating organisms (PAOs) in all systems were stable and showed little variability between each plant, while glycogen accumulating organisms (GAOs) were present in two of the plants. The metabolic activity of each sludge showed the frequent involvement of the anaerobic tricarboxylic acid cycle (TCA) in PAO metabolism for the anaerobic generation of reducing equivalents, in addition to the more frequently reported glycolysis pathway. Metabolic variability in the use of the two pathways was also observed, between different systems and in the same system over time. The metabolic dynamics was linked to the availability of glycogen, where a higher utilisation of the glycolysis pathway was observed in the two systems employing side-stream hydrolysis, and the TCA cycle was more active in the A(2)O systems. Full-scale plants that showed higher glycolysis activity also exhibited superior P removal performance, suggesting that promotion of the glycolysis pathway over the TCA cycle could be beneficial towards the optimisation of EBPR systems.