The study developed an autotrophic synthetic medium for enriching anaerobic ammonium-oxidizing (Anammox) microorganisms in a fluidized bed reactor (FBR). The medium contained ammonium and nitrite as electron donors and acceptors, respectively, with carbonate as the sole carbon source. Preliminary studies indicated that nitrite and the absence of organic electron donors were essential for Anammox activity. In the FBR, the conversion rate of the enrichment culture was 3 kg NH4 m-3 d-1 when fed with 30 mM NH4, equivalent to a specific anaerobic ammonium oxidation rate of 1000-1100 nmol NH4 h-1 (mg volatile solids)-1. The maximum specific oxidation rate was 1500 nmol NH4 h-1 (mg volatile solids)-1. Per mol NH4 oxidized, 0.041 mol CO2 were incorporated, resulting in an estimated growth rate of 0.001 h-1. The main product of the Anammox reaction was N2, but about 10% of the N-feed was converted to NO2. The overall nitrogen balance showed a ratio of NH4-conversion to NO2-conversion and NO2-production of 1:1:31 ± 0.06: 0.22 ± 0.02. Acetylene, phosphate, and oxygen were found to be strong inhibitors of Anammox activity. The dominant microorganism in the enrichment culture had an irregular morphology and increased in ether lipids during enrichment, changing from brownish to red. Cytochrome spectra showed a peak at 470 nm gradually increasing in intensity during enrichment.The study developed an autotrophic synthetic medium for enriching anaerobic ammonium-oxidizing (Anammox) microorganisms in a fluidized bed reactor (FBR). The medium contained ammonium and nitrite as electron donors and acceptors, respectively, with carbonate as the sole carbon source. Preliminary studies indicated that nitrite and the absence of organic electron donors were essential for Anammox activity. In the FBR, the conversion rate of the enrichment culture was 3 kg NH4 m-3 d-1 when fed with 30 mM NH4, equivalent to a specific anaerobic ammonium oxidation rate of 1000-1100 nmol NH4 h-1 (mg volatile solids)-1. The maximum specific oxidation rate was 1500 nmol NH4 h-1 (mg volatile solids)-1. Per mol NH4 oxidized, 0.041 mol CO2 were incorporated, resulting in an estimated growth rate of 0.001 h-1. The main product of the Anammox reaction was N2, but about 10% of the N-feed was converted to NO2. The overall nitrogen balance showed a ratio of NH4-conversion to NO2-conversion and NO2-production of 1:1:31 ± 0.06: 0.22 ± 0.02. Acetylene, phosphate, and oxygen were found to be strong inhibitors of Anammox activity. The dominant microorganism in the enrichment culture had an irregular morphology and increased in ether lipids during enrichment, changing from brownish to red. Cytochrome spectra showed a peak at 470 nm gradually increasing in intensity during enrichment.