AbstractThe reaction rates (r and r) in the two‐step nitrification reaction were measured in a fluidized‐sand‐bed biofilm reactor under a range of steady‐state conditions with respect to bulk NH, NO, and O2 concentrations. It was shown from theory and experiment that under low NH concentration conditions, if the O2/NH concentration ratio in the bulk liquid is less than the stoichiometric coefficient (3.4 mg/mg), then oxygen will be rate limiting. In all experiments r decreased more than r under low oxygen conditions. This resulted in high NO effluent concentrations under low residence time conditions. The influence of the oxygen penetration effects on the relative values of r and r was experimentally shown to be caused either by the Nitrobacter location in the inner biofilm regions or by a Km effect for oxygen. Theoretical support of these findings was provided by a differential diffusion‐reaction model which was used to simulate the experimental results.