Nitrite has been found in previous research an inhibitor on anoxic phosphorus uptake in enhanced biological phosphorus removal systems (EBPR). However, the inhibiting nitrite concentration reported varied in a large range. This study investigates the nitrite inhibition on anoxic phosphorus uptake by using four different mixed cultures performing EBPR with pH considered an important factor. The results showed that the protonated species of nitrite, HNO2 (or free nitrous acid, FNA), rather than nitrite, is likely the actual inhibitor on the anoxic phosphorus uptake, as revealed by the much stronger correlation of the phosphorus uptake rate with the FNA than with the nitrite concentration. All the four EBPR sludges showed decreased anoxic phosphorus uptake rates with increased FNA concentrations in the studied range of 0.002–0.02 mg HNO2-N/L. The phosphorus uptake by all four cultures was completely inhibited at 0.02 mg HNO2-N/L. Granular sludge appeared to be more tolerant to HNO2 than flocular sludge likely due to its stronger resistance to the transfer of nitrite into the bacterial aggregates. Furthermore, denitrification by the phosphorus-accumulating organisms (PAOs) was also found to be inhibited by HNO2. The denitrification rate decreased by approximately 40% when the FNA concentration was increased from 0.002 to 0.02 mg HNO2-N/L. Biotechnol. Bioeng. 2007;98: 903–912. © 2007 Wiley Periodicals, Inc.