Iron(III) profiles of flooded paddy soil incubated in the greenhouse indicated oxidation of iron(II) in the upper 6 mm soil layer. Measurement of oxygen with a Clark-type microelectrode showed that oxygen was only responsible for the oxidation of iron(II) in the upper 3 mm. In the soil beneath, nitrate could be used as electron acceptor instead of oxygen for the oxidation of the iron(II). Nitrate was still available 3 mm below the soil surface, and denitrifying activity was indicated by higher concentrations of nitrite between 3 and 6 mm soil depth. Nitrate was generated by nitrification from ammonium. Ammonium concentrations increased beneath 6 mm soil depth, indicating ammonium release and diffusion from deeper soil layers. High concentrations of ammonium were also found at the surface, probably resulting from N2 fixation by cyanobacteria. Experimental adjustment of the nitrate concentration in the flooding water to 200 microM stimulated nitrate-dependent iron(II) oxidation, which was indicated by significantly lower iron(II) concentrations in soil layers in which nitrate-dependent iron(II) oxidation was proposed. Soil incubated in the dark showed high iron(III) concentrations only in the layer where oxygen was still available. In this soil, the nitrogen pool was depleted because of the lack of N2 fixation by cyanobacteria. In contrast, soil incubated in the dark with 500 microM nitrate in the flooding water showed significantly higher iron(II) and significantly lower iron(II) concentrations in the anoxic soil layers, indicating nitrate-dependent iron(II) oxidation. Anoxic incubations of soil with nitrate in the flooding water also showed high concentrations of iron(II) and low concentrations of iron(II) in the upper 3 mm. As oxygen was excluded in anoxic incubations, the high iron(III) concentrations are a sign of the activity of nitrate-dependent iron(II) oxidizers. The presence of these bacteria in non-amended soil was also indicated by the most probable number (MPN) counts of nitrate-dependent iron(II) oxidizers in the layer of 3-4 mm soil depth, which revealed 1.6 x 10(6) bacteria g(-1) dry weight.