High autotrophic nitrogen removal rates of 858mg NL(-1) day(-1) or 1.55g Nm(-2) day(-1) were obtained in a lab-scale rotating biological contactor treating an ammonium rich influent. It was postulated that ammonium was removed as dinitrogen gas by a sequence of aerobic ammonium oxidation to nitrite taking place in the outer biofilm layer and anaerobic ammonium oxidation with nitrite as electron acceptor occuring in the deeper biofilm layer. Chemical evidence for anaerobic ammonium oxidation within intact biofilm sludge from a lab-scale rotating biological contactor could be provided, without direct identification of responsible organisms catalysing this reaction. 15N tracer techniques were used for identification and quantification of nitrogen transformations. In batch tests with biofilm sludge at dissolved oxygen concentrations lower than 0.1mgL(-1), ammonium and nitrite did react in a stoichiometric ratio of 1:1.43 thereby forming dinitrogen. 15N isotope dilution calculations revealed that anaerobic ammonium oxidation was the major nitrogen transformation leading to concomitant ammonium and nitrite removal. Isotopic analysis of the produced biogas showed that both ammonium-N and nitrite-N were incorporated in N(2).