Bacterial community structure and the predominant nitrifying activities and populations in each compartment of a three-compartment activated sludge system were determined. Each compartment was originally inoculated with the same activated sludge community entrapped in polyethylene glycol gel granules, and ammonium nitrogen was supplied to the system in an inorganic salts solution at a rate of 5.0 g of N liter of granular activated sludge¹ day¹. After 150 days of operation, the system was found to comprise a series of sequential nitrifying reactions (K. Noto, T. Ogasawara, Y. Suwa, and T. Sumino, Water Res. 32:769-773, 1998), presumably mediated by different bacterial populations. Activity data showed that all NH₄-N was completely oxidized in compartments one and two (approximately half in each), but no significant nitrite oxidation was observed in these compartments. In contrast, all available nitrite was oxidized to nitrate in compartment three. To study the microbial populations and communities in this system, total bacterial DNA isolated from each compartment was analyzed for community structure based on the G+C contents of the component populations. Compartment one showed dominant populations having 50 and 67% G+C contents. Compartment two was similar in structure to compartment one. The bacterial community in compartment three had dominant populations with 62 and 67% G+C contents and retained the 50% G+C content population only at a greatly diminished level. The 50% G+C content population from compartment one hybridized strongly with amo (ammonia monooxygenase) and hao (hydroxylamine oxidoreductase) gene probes from Nitrosomonas europaea. However, the 50% G+C content population from compartment two hybridized strongly with the hao probe but only weakly with the amo probe, suggesting that the predominant ammonia-oxidizing populations in compartments one and two might be different. Since different activities and populations come to dominate in each compartment from an identical inoculum, it appears that the nitrification processes may be somewhat incompatible, resulting in a series of sequential reactions and different communities in this three-compartment system.