The effect of the overlying water velocity on ammonium (NH4+) uptake by benthic biofilms was studied in a recirculating laboratory flume (260 cm long, 29 cm wide), packed with 5 cm of silica sand arranged into bedforms. NH4+ uptake was determined as the reduction in NH4+ concentration in the water at average overlying water velocities of 0.8, 2, 4 and 8 cm s−1. NH4+ uptake was relatively constant under laminar flow conditions but increased when the flow regime became turbulent (>4 cm s−1). This pattern was observed for two biofilms differing in their total biomass and in the abundance of the ammonia-oxidizing bacteria, thus indicating that NH4+ uptake was strongly controlled by mass-transfer processes. The near stoichiometric relationship between the rates of NH4+ uptake and nitrate (NO3−) accumulation suggests that aerobic nitrification was the main route for NH4+ uptake. Microelectrode measurements showed a sharp decline of oxygen concentrations and pH values within the biofilms, thus supporting strong nitrification activity within the surficial section of the benthic biofilms. The results of this study highlight the key role of hydrodynamic conditions in regulating NH4+ uptake in the transition from laminar to turbulent flow conditions. Copyright © 2012 John Wiley & Sons, Ltd.