Understanding the nature and fate of nitrogen (N) in freshwater systems is crucial for assessing the risk of eutrophication. However, there is a paucity of information on the characterization of fluvial N in upland peat-dominated environments. Here, we employ a combination of field sampling and tangential flow ultrafiltration (TFU) to investigate the concentrations and fluxes of low molecular weight (LMW) and high molecular weight (HMW) dissolved organic N (DON) in a peatland stream-reservoir system in the south Pennines (UK). Our TFU results show that ∼26% of DON concentration is LMW DON and represents an estimated fluvial flux of 3.07 ± 22 kg N ha-1 during the study period. Our mass balance results reveal that the reservoir retains 71% of LMW DON input, which accounts for ∼25% retention of bioavailable (dissolved inorganic N + LMW DON) N. Our study suggests that current understanding of inorganic N as the sole source of bioavailable N with eutrophic significance in upland freshwaters requires a reappraisal. Evaluation of ecosystem response to increased loading of N needs to include a consideration of LMW DON.