Partial nitrification combined with Anammox in a single reactor (the CANON process) is an energy-efficient N-removal technology that could substantially lower the N-load of a WWTP by separate treatment of nitrogen-rich side streams, preventing the need for extensive expansion and reducing the total energy requirement. This study looks at the enrichment of Anammox from activated sludge and its application in the CANON process on lab-scale. The aim was to identify the critical process control parameters necessary for successful operation of CANON. An Anammox culture capable of removing 0.6 kg N/m3/d was enriched in 14 weeks in a sequencing batch reactor. Nitrifying biomass was inoculated into the Anammox reactor (10% v/v) together with limited oxygen supply (< 8 mL/min) to initiate the CANON process in continuous culture. The small flocs formed by the biomass (< 1000 microm) were sensitive to low O2 concentrations (< 0.1 mg/L) which prevented simultaneous nitrification and Anammox. Operation with 20 min aerobiosis and 30 min anaerobiosis was necessary to achieve sustained, completely autotrophic N-removal for an extended period at a rate of 0.08 kg N/m3/d. Essential process control parameters for stable CANON operation were the nitrite concentration, oxygen concentration, pH and the temperature.