Spatial microbial distributions of nitrifiers and heterotrophs in undefined mixed-population biofilms were experimentally investigated using a microslicer technique and correlated with nitrification efficiency of the biofilm system. The general stratification of different bacterial groups in the biofilm was simulated using a one-dimensional (1-D) mathematical biofilm accumulation model (BAM) and compared with the experimental results. Biofilms were cultured at three C:N ratios of feed solutions In a partially submerged rotating biological contactor (RBC). It was shown that the biofilms were vertically stratified (from biofilm surface to substratum). At C:N = 0, heterotrophs and nitrifiers coexisted in the outermost biofilm and heterotrophs dominated in the innermost biofilm. At C:N = 1.5, heterotrophs outcompeted nitrifiers for dissolved oxygen and space; thus, heterotrophs dominated in the outermost biofilm and nitrifiers were present only in the deeper biofilm. Nitrifiers and heterotrophs coexisted in the innermost biofilm. An increase in the influent C:N ratio resulted in stronger stratification of microbial species, as well as inhibition of nitrification. In batch experiments, NH4-N utilization rate (R(NH4-N)) was almost the same at each substrate C : N ratio even though NH, oxidizers were predominantly present in the deeper biofilm. The biofilm performance could not be sufficiently explained by the obtained microbial spatial distribution, suggesting that one-dimensional description of microbial distribution was not good enough and three-dimensional measurements of microbial spatial distribution is necessary. Total bacterial densities increased by a factor of 3-17 with biofilm depth. The metabolically active cell fraction decreased from 35 +/- 13% in the outermost biofilm to 15 +/- 4% in the innermost biofilm, presumably due to substrate limitation. The model predicted more pronounced stratification of nitrifiers and heterotrophs than the observed results. This discrepancy could be attributed to the real biofilms that were structurally heterogeneous (e.g., water channels), which could not be described by the one-dimensional model. The results of this study clearly indicate the limitation of 1-D biofilm models to describe the extent of stratification of nitrifiers and heterotrophs and suggest a 3-D model is necessary.