In this study, the biomass fractions of heterotrophs (X H ) and phosphate-accumulating organisms (X P A O ) were estimated through experimental results and theoretical calculation on the mass balance of organic matter, nitrogen and phosphorus in a pilot scale biological nutrient removal process. The process, comprised of conventional anaerobic-anoxic-aerobic activated sludge and a rotating biological contactor in the aerobic stage, was controlled under steady state under conditions of a solid retention time of 10 days and a dissolved oxygen concentration of 2.0<space>mg/l in the aerobic stage. For long term operation, the process exhibited high performance in organic matter, nitrogen and phosphorus removal. The removal efficiency was 96.5, 70 and 100% of organic matter, total nitrogen and total phosphorus, respectively. According to the mass balance calculation, approximately 62.0% of organic matter, 48.0% of nitrogen and 47.2% of phosphorus of influent were removed in the form of carbon dioxide, nitrogen gas and polyphosphate, respectively. Regarding X H and X P A O as the major organisms responsible for biomass production, the yield fractions of X H growth in anoxic and aerobic conditions were 48.4 and 39.4%, respectively. Moreover, the yield fraction of X P A O growth in aerobic conditions was 12.2%. These results showed that X H was the major community; moreover, only a minute amount of X P A O was present in this nitrogen and phosphorus removal system for municipal wastewater treatment.