The microzonation of O₂ respiration, H₂S oxidation, and SO₄^2- reduction in aerobic trickling-filter biofilms was studied by measuring concentration profiles at high spatial resolution (25 to 100 μm) with microsensors for O₂, S^2-, and pH. Specific reaction rates were calculated from measured concentration profiles by using a simple one-dimensional diffusion reaction model. The importance of electron acceptor and electron donor availability for the microzonation of respiratory processes and their reaction rates was investigated. Oxygen respiration was found in the upper 0.2 to 0.4 mm of the biofilm, whereas sulfate reduction occurred in deeper, anoxic parts of the biofilm. Sulfate reduction accounted for up to 50% of the total mineralization of organic carbon in the biofilms. All H₂S produced from sulfate reduction was reoxidized by O₂ in a narrow reaction zone, and no H₂S escaped to the overlying water. Turnover times of H₂S and O₂ in the reaction zone were only a few seconds owing to rapid bacterial H₂S oxidation. Anaerobic H₂S oxidation with NO₃^- could be induced by addition of nitrate to the medium. Total sulfate reduction rates increased when the availability of SO₄^2- or organic substrate increased as a result of deepening of the sulfate reduction zone or an increase in the sulfate reduction intensity, respectively.