The application of nitrogen (N) fertilizer to agricultural fields is important for maximizing productivity; however, excess inputs of N can impair downstream waters. This excess N can increase denitrification rates, wherein nitrate (NO3--N) is converted to dinitrogen (N2) gas, permanently removing bioreactive N from the ecosystem. Increased N fertilizer use and warming temperatures driven by climate change may further increase denitrification by increasing both NO3--N concentrations and biological activity. Studies often focus on denitrification in headwater streams; yet, larger rivers are known to play a major role in reach-scale N retention. Moreover, it remains uncertain how denitrification rates differ across seasons and stream sizes, as well as how efficiently this permanent N sink reduces N loads to downstream waterways. To estimate reach-scale denitrification, we conducted three seasonal diel sampling events over 36h in an agriculturally-dominated mainstem river (Tippecanoe R.; TIP) in Indiana and its tributary (Shatto Ditch; SHA) using the open-channel N2-exchange method. Our preliminary results show that reach-scale denitrification was ~4X higher in the tributary than the mainstem in both summer (SHA=75.4 mg N m-2 h-1, TIP=18.9 mg N m-2 h-1) and fall (SHA=50.6 mg N m-2 h-1, TIP=13.5 mg N m-2 h-1). We also found that denitrification from SHA and TIP was related to NO3--N availability (Pearson; r=0.99), where NO3--N concentration was higher in SHA than TIP in both summer (SHA=4.5 mg N L-1, TIP=1.0 mg N L-1) and fall (SHA=2.8 mg N L-1, TIP=1.0 mg N L-1). This work expands our knowledge of denitrification in fluvial ecosystems and improves our understanding of how human-impacted freshwaters of all sizes are reducing N loads to downstream aquatic ecosystems.