In this study, the effect of different operational conditions on biofilm development and nitrification in three moving-bed biofilm reactors (MBBRs) was investigated: two reactors were operated in a continuously fed regime and one in sequencing-batch mode. The presence of organic carbon reduced the time required to form stable nitrifying biofilms. Subsequent stepwise reduction of influent COD caused a decrease in total polysaccharide and protein content, which was accompanied by a fragmentation of the biofilm, as shown by scanning electron microscopy, and by an enrichment of the biofilm for nitrifiers, as observed by fluorescent in situ hybridization (FISH) analysis. Polysaccharide and protein concentrations proved to be good indicators of biomass development and detachment in MBBR systems. Ammonium- and nitrite-oxidizing bacteria activities were affected when a pulse feeding of 4 g of NH 4-N/(m 2·day) was applied. Free nitrous acid and free ammonia were likely the inhibitors for ammonium- and nitrite-oxidizing bacteria. © 2012 American Chemical Society.