The microbiology and the feasibility of a new, single-stage, reactor for completely autotrophic ammonia removal were investigated. The reactor was started anoxically after inoculation with biomass from a reactor performing anaerobic ammonia oxidation (Anammox). Subsequently, oxygen was supplied to the reactor and a nitrifying population developed. Oxygen was kept as the limiting factor. The development of a nitrifying population was monitored by Fluorescence In Situ Hybridization and off-line activity measurements. These methods also showed that during steady state, anaerobic ammonium-oxidizing bacteria remained present and active. In the reactor, no aerobic nitrite-oxidizers were detected. The denitrifying potential of the biomass was below the detection limit. Ammonia was mainly converted to N2 (85%) and the remainder (15%) was recovered as NO3-. N2O production was negligible (less than 0.1%). Addition of an external carbon source was not needed to realize the autotrophic denitrification to N2.