A simple, one reactor vessel system, called a fill-and-draw system, was developed for the study of enhanced biological phosphate removal under defined conditions. Sludge was grown in a medium with acetate and glucose as sole energy and carbon sources. The sludge was exposed to cycles with three distinct, consecutive periods; first an anaerobic period, then an aerobic period and finally a settlement period. In the period of settlement one third of the liquid was replaced with fresh medium. Sludge grown under this regime became considerably enriched with polyphosphate-accumulating bacteria. The polyphosphate content reached up to 110 mg P/g dry weight. The amount of polyphosphate in the cells during steady state depended on the acetate:glucose ratio, the nitrate and phosphate concentration in the medium. Highest phosphate accumulation was obtained with an acetate:glucose ratio of 9:1. Intracellular polyphosphate was formed during the aerobic period and was anaerobically hydrolysed and released as phosphate into the medium. In the absence of oxygen and in the presence of 2 g acetate-COD/1, 80-90% of phosphate was released by sludge containing 100 mg P/g dry weight. In the absence of acetate only 2-19% of the accumulated phosphate was excreted.