We conducted an in-stream tracer experiment with Br and ¹⁵N-enriched NO₃^- to determine the rates of denitrification and related processes in a gaining NO₃^- -rich stream in an agricultural watershed in the upper Mississippi basin in September 2001. We determined reach-averaged rates of N fluxes and reactions from isotopic analyses of NO₃^-, NO₂^-, N₂, and suspended particulate N in conjunction with other data in a 1.2-km reach by using a forward time-stepping numerical simulation that included groundwater discharge, denitrification, nitrification, assimilation, and air-water gas exchange with changing temperature. Denitrification was indicated by a systematic downstream increase in the d15N values of dissolved N₂. The reach-averaged rate of denitrification of surface-water NO₃^- indicated by the isotope tracer was approximately 120 ± 20 µmol m^-2 h^-1 (corresponding to zero- and first-order rate constants of 0.63 µmol L^-1 h^-1 and 0.009 h^-1, respectively). The overall rate of NO₃^- loss by processes other than denitrification (between 0 and about 200 µmol m^-2 h^-1) probably was less than the denitrification rate but had a large relative uncertainty because the NO₃^- load was large and was increasing through the reach. The rates of denitrification and other losses would have been sufficient to reduce the stream NO₃^- load substantially in the absence of NO₃^- sources, but the losses were more than offset by nitrification and groundwater NO₃^- inputs at a combined rate of about 500-700 µmol m^-2 h^-1. Despite the importance of denitrification, the overall mass fluxes of N₂ were dominated by discharge of denitrified groundwater and air-water gas exchange in response to changing temperature, whereas the flux of N₂ attributed to denitrification was relatively small. The in-stream isotope tracer experiment provided a sensitive direct reach-scale measurement of denitrification and related processes in a NO₃^- -rich stream where other mass-balance methods were not suitable because of insufficient sensitivity or offsetting sources and sinks. Despite the increasing NO₃^- load in the experimental reach, the isotope tracer data indicate that denitrification was a substantial permanent sink for N leaving this agricultural watershed during low-flow conditions.