This study evaluates the community structure in nitrifying granules (average diameter of 1600 mu m) produced in an aerobic reactor fed with ammonia as the sole energy source by a multivalent approach combining molecular techniques, microelectrode measurements and mathematical modelling. Fluorescence in situ hybridization revealed that ammonia-oxidizing bacteria dominated within the first 200 mu m below the granule surface, nitrite-oxidizing bacteria a deeper layer between 200 and 300 mu m, while heterotrophic bacteria were present in the core of the nitrifying granule. Presence of these groups also became evident from a 16S rRNA clone library. Microprofiles of NH4+, NO2-, NO3- and O-2 concentrations measured with microelectrodes showed good agreement with the spatial organization of nitrifying bacteria. One- and two-dimensional numerical biofilm models were constructed to explain the observed granule development as a result of the multiple bacteria-substrate interactions. The interaction between nitrifying and heterotrophic bacteria was evaluated by assuming three types of heterotrophic bacterial growth on soluble microbial products from nitrifying bacteria. The models described well the bacterial distribution obtained by fluorescence in situ hybridization analysis, as well as the measured oxygen, nitrite, nitrate and ammonium concentration profiles. Results of this study are important because they show that a combination of simulation and experimental techniques can better explain the interaction between nitrifying bacteria and heterotrophic bacteria in the granules than individual approaches alone.