Microprofiles of O2 and NO3- were measured in nitrifying biofilms from the trickling filter of an aquaculture water recirculation system. By use of a newly developed biosensor for NO3-, it was possible to avoid conventional interference from other ions. Nitrification was restricted to a narrow zone of 50 microns on the very top of the film. In the same biofilms, the vertical distributions of members of the lithoautotrophic ammonia-oxidizing genus Nitrosomonas and of the nitrite-oxidizing genus Nitrobacter were investigated by applying fluorescence in situ hybridization of whole fixed cells with 16S rRNA-targeted oligonucleotide probes in combination with confocal laser-scanning microscopy. Ammonia oxidizers formed a dense layer of cell clusters in the upper part of the biofilm, whereas the nitrite oxidizers showed less-dense aggregates in close vicinity to the Nitrosomonas clusters. Both species were not restricted to the oxic zone of the biofilm but were also detected in substantially lower numbers in the anoxic layers and even occasionally at the bottom of the biofilm.