This study combined bacterial incubation and hydrogeochemical methods to investigate denitrification in the surface water, top soil (0–20 cm), and shallow groundwater of the Ochi wetland in Japan. Surface water was rich in nitrate (40 mg/l) and denitrifying bacteria (700 per ml). Three functional zones in the wetland were identified in the top soil and shallow groundwater. In the upstream portion of the wetland (Zone I), the counts of denitrifying bacteria in top soil increased from 5200 to 14 970 per ml and nitrate decreased from 25·4 to 1·8 mg/l. Organic carbon concentrations decreased as sulfate increased from 4·0 to 9·6 mg/l in this zone. In the middle-stream of the wetland (Zone II), all concentrations of major anions, iron, organic carbon, and total nitrogen content in top soil were relatively constant, but the counts of denitrifying bacteria increased up to 70 200 per ml. In the downstream portion of the wetland (Zone III), complete removal of nitrate resulted in sharp reduction of counts of denitrifying bacteria. Correspondingly, dissolved oxygen (DO), organic carbon, and total nitrogen increased in this zone. Counts of denitrifying bacteria were lower in shallow groundwater than in top soil; nitrate concentrations in shallow groundwater were also very low in this zone. DO and the oxidation/reduction potential data suggest that groundwater flows to the surface along an extended flow path, thus providing nitrate for the denitrifying community. Copyright © 2011 John Wiley & Sons, Ltd.