The development of stream biofilms was studied using a rotating annular biofilm reactor system for cultivation and confocal laser scanning microscopy for structural examination. Biofilms were generated by feeding the reactor with raw river water as inoculum and a sole nutrient source. The biofilm developed during 56 days from microcolonies via a thin patchy film into a ridged structure. The ridges were oriented parallel to the direction of flow. The parallel ridges varied from a few microns up to 500 μm thick and consisted of cellular and non-cellular material. The architecture of the film was in contrast to previous views of a homogeneous biofilm structure and more recent biofilm models with channels and inverted biomass distribution. The difference may be attributed to the presence and co-adsorption of humic and detrital material in natural lotic systems. This material building the ridges supplied an increased surface area for colonization and may serve as an additional source of nutrients. The biofilms on the removable slides showed a thickness gradient across the width and the length of the slides which may be caused by the flow pattern of the water inside the reactor. By using a live/dead fluorescent probe the population heterogeneity of the biofilm was demonstrated in space and time. In addition, the dominance of certain bacteria and their extracellular polymeric products was examined using lectin binding analyses. The rotating annular biofilm reactor may be a useful tool for morphological studies of complex microbial films, particularly those developing under turbulent flow regimes which are typical for aqueous environmental ecosystems.