A membrane-aerated biofilm reactor (MABR) capable of simultaneous nitrification and denitrification in a single reactor vessel was developed to investigate the characteristics of nitrogen removal from high-strength nitrogenous wastewater, and biofilm analysis using microelectrodes and the fluorescence in situ hybridization (FISH) technique was performed. Mean removal percentages of total organic carbon (TOC) and nitrogen were 96% and 83% at removal rates of 5.76 g-C m(-2) d(-1) and 4.48 g-N m(-2) d(-1), respectively. For stable removal efficiency, constant washing of the biofilm was needed. Dissolved oxygen microelectrode measurement revealed that the biofilm thickness was about 1600 mum, and that oxygen penetrated about 300 to 700 mum from the outer surface of the membrane. Furthermore, FISH analysis revealed that ammonia-oxidizing bacteria (AOB) were located near the outer surface of the membrane, whereas other bacteria were located from the inner to the outer part of the biofilm. Combining these results demonstrated that simultaneous nitrification and denitrification occurred in the biofilm of the MABR system. In addition, stoichiometric analysis revealed that after 130 d the free ammonia (FA) concentration ranged within the concentration causing inhibition of the growth of nitrite oxidizing bacteria (NOB) and that AOB consumed 86% of the oxygen supplied through the intra-membrane. These results indicate that nitrogen removal not via nitrate but via nitrite was mainly achieved in the MABR system.