The effect of water velocity on nitrification rates in fixed bed biofilters was investigated in three freshwater pilot scale RAS with rainbow trout. Removal of total ammonia nitrogen (TAN) and nitrite-nitrogen were assessed by NH4Cl spikes and tested at four different water velocities in the biofilters (1.4, 5.4, 10.8 and 16.2 m h− 1) under identical conditions. Water velocities below 10.8 m h− 1 significantly reduced TAN- and nitrite removal rates. The surface specific TAN removal rates correlated with the TAN concentrations at the water velocities 10.8 and 16.2 m h− 1, and the first order surface removal rate constant was estimated at 0.45 m h− 1. However, no correlations between TAN removal and TAN concentrations were found at the lowest velocities. Up to five-fold elevated nitrite levels were found in the RAS when biofilters were operated at 1.4 m h− 1 compared to the trials at other water velocities, substantiating the significant effect of water velocity on both nitrification processes. The importance of biofilter hydraulics documented in this pilot scale RAS probably have implications for design and operation in larger scale RAS.