Two laboratory-scale sequencing batch reactors (SBRs) were operated for enhanced biological phosphorus removal (EBPR) in alternating anaerobic–aerobic or alternating anaerobic–anoxic modes, respectively. Polyphosphate-accumulating organisms (PAOs) were enriched in the anaerobic–aerobic SBR and denitrifying PAOs (DPAOs) were enriched in the anaerobic–aerobic SBR. Fluorescence in situ hybridization (FISH) demonstrated that the well-known PAO, “Candidatus Accumulibacter phosphatis” was abundant in both SBRs, and post-FISH chemical staining with 4,6-diamidino-2-phenylindol (DAPI) confirmed that they accumulated polyphosphate. When the anaerobic–anoxic SBR enriched for DPAOs was converted to anaerobic–aerobic operation, aerobic uptake of phosphorus by the resident microbial community occurred immediately. However, when the anaerobic–aerobic SBR enriched for PAOs was exposed to one cycle with anoxic rather than aerobic conditions, a 5-h lag period elapsed before phosphorus uptake proceeded. This anoxic phosphorus-uptake lag phase was not observed in the subsequent anaerobic–aerobic cycle. These results demonstrate that the PAOs that dominated the anaerobic–aerobic SBR biomass were the same organisms as the DPAOs enriched under anaerobic–anoxic conditions. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 140–148, 2003.