Microelectrodes for O-2 and NO2-/NO3- and fluorescently labelled 16S rRNA-targeted oligonucleotide probes were combined to examine the activity and stratification of nitrifying bacteria in a trickling filter biofilm, Microprofiles showed that O-2 consumption and NO3-/NO2- production were restricted to the upper 50-100 mu m of the biofilm, The vertical distribution of the nitrifying bacteria Nitrosomonas sp. and Nitrobacter sp. was investigated by fluorescent in situ hybridisation (FISH) with specific oligonucleotides. Nitrifiers formed a dense layer of cells and cell clusters in the upper part of the biofilm, This correlates well with the measured activity profiles, Ammonia-and nitrite-oxidisers occurred in close vicinity to each other supporting a fast sequential metabolism from ammonia to nitrate. Both species were not restricted to the oxic part of the biofilm, but also appeared -in lower numbers-in the anoxic layers on the bottom of the biofilm, A short term decrease in the O-2 concentration of the bulk water resulted in a quick decrease in O-2 penetration and metabolic rates inside the biofilm. However, neither the stratification nor the cellular ribosome content of nitrifiers changed within a few hours. (C) 1997 IAWQ. Published by Elsevier Science Ltd.