The contribution of the biochemical pathways nitrification, denitrification, and dissimilatory NO3− reduction to NH4+ (DNRA) to the accumulation of NO2− in freshwaters is governed by the species compositions of the bacterial populations resident in the sediments, available carbon (C) and nitrogen (N) substrates, and environmental conditions. Recent studies of major rivers in Northern Ireland have shown that high NO2− concentrations found in summer, under warm, slow-flowing conditions, arise from anaerobic NO3− reduction. Locally, agricultural pollutants entering rivers are important C and N sources, providing ideal substrates for the aquatic bacteria involved in cycling of N. In this study a range of organic C compounds commonly found in agricultural pollutants were provided as energy sources in 48-h incubation experiments to investigate if the chemical compositions of the pollutants affected which NO3− reduction pathway was followed and influenced subsequent NO2− accumulation. Carbon stored within the sediments was sufficient to support DNRA and denitrifier populations, and the resulting NO2− peak (80 μg of N liter−1 [approximate]) observed at 24 h was indicative of the simultaneous activities of both bacterial groups. The value of glycine as an energy source for denitrification or DNRA appeared to be limited, but glycine was an important source of additional N. Glucose was an efficient substrate for both the denitrification and DNRA pathways, with a NO2− peak of 160 μg of N liter−1 noted at 24 h. Addition of formate and acetate stimulated continuous NO2− production throughout the 48-h period, caused by partial inhibition of the denitrification pathway. The formate treatment resulted in a high NO2− accumulation (1,300 μg of N liter−1 [approximate]), and acetate treatment resulted in a low NO2− concentration (<100 μg of N liter−1).