Rivers are important sources of N2O emissions into the atmosphere. Nevertheless, N2O production processes in rivers are not well identified. We measured concentrations and isotopic ratios of N2O, NH4?, NO2-, and NO3- in surface water to identify the microbial processes of N2O production along the Tama River in Japan. We also measured the functional gene abundance of nitrifiers and denitrifiers (amoAbacteria, nirK, nirS, nosZ clade I, nosZ clade II) together with concentrations of dissolved organic carbon (DOC) and fluorescence intensities of protein and humic components of dissolved organicmatter (DOM) to support the elucidation of N2O production processes. The observed nitrogen (d15N) and oxygen (d18O) of N2O were within the expected isotopic range of N2O produced by nitrate reduction, indicating that N2O was dominantly produced by denitrification. The positive significant correlation between N2ONet concentration and nirK gene abundance implied that nitrifiers and denitrifiers are contributors to N2O production. Fluorescence intensities of protein and humic components of DOM and concentrations of DOC did not show significant correlations with N2O concentrations, which suggests that DOC and abundance of DOM components do not control dissolved N2O. Measurement of isotope ratios of N2O and its substrates was found to be a useful tool to obtain evidence of denitrification as the main source of N2O production along the Tama River.