Ammonium is a substrate of the anaerobic ammonium oxidation (Anammox) process but it has been suggested as a substrate-inhibitor because of the action of its unionized form, free ammonia. High pH of the medium is also an important limiting factor of the Anammox bacteria. Both effects are difficult to discriminate. In this work the inhibitory effects of high pH, total ammonia (TA) and NH3 on the Anammox process were investigated simultaneously. Results confirmed that TA caused no inhibition and high pH is a much more important inhibiting factor than NH3 in mildly alkaline conditions, based on a multi-factorial analysis. Values of pH higher than 7.6 caused Anammox inhibition > 10 % and should be avoided during the application of the Anammox process in practice.