The effect of nitrate on phosphorus release by biological phosphorus removing organisms has been studied. Denitrifying (DPB) or aerobic phosphorus removing bacteria were enriched in an anaerobic-anoxic or anaerobic-aerobic sequencing batch reactor (SBR). The enrichment sludges were used in batch tests, in which the effect of simultaneous presence of substrate (HAc) and nitrate was studied on the phosphorus release. It could be concluded that a reduction of the phosphorus release by nitrate in biological phosphorus removal systems is partly due to the presence of DPB, which utilize HAc for denitrification, not for phosphorus release. PHB (poly-β-hydroxybutyrate) was always produced and phosphorus was released by DPB sludge when nitrate and HAc were simultaneously present. The reducing power (NADH 2 ) and the energy (ATP) for this process seemed to be obtained from HAc oxidation by nitrate as well as from polyphosphate degradation. After removal of the HAc, PHB degradation and phosphorus uptake occurred.