The Potomac River is a large source of N to Chesapeake Bay, where reducing nutrient loads is a focus of efforts to improve trophic status. Better understanding of NO₃^– loss, reflected in part by diel variation in NO₃^– concentrations, may refine model predictions of N loads to the Bay. We analyzed 2 y of high-frequency NO₃^– sensor data in the Potomac to quantify seasonal variation in the magnitude and timing of diel NO₃^– loss. Diel patterns were evident, especially during low flow, despite broad seasonal and flow-driven variation in NO₃^– concentrations. Diel variation was ~0.01 mg N/L in winter and 0.02 to 0.03 mg N/L in summer with intermediate values in spring and autumn, equivalent to <1% of the daily mean NO₃^– concentration in winter and ~2 to 4% in summer. Maximum diel NO₃^– values generally occurred in mid- to late morning, with more repeatable patterns in summer and wider variation in autumn and winter. Diel NO₃^– loss reduced loads by 0.7% in winter and 3% in summer. These losses were less than estimates of total in-stream NO₃^– load loss across the basin that averaged 33% of the annual groundwater contribution to the river. Water temperature and discharge had stronger relationships to the daily magnitude of diel NO₃^– variation than did photosynthetically active radiation. Estimated diel areal NO₃^– loss rates were generally >1000 mg N m^–2 d^–1, greater than most published values because measurements in this large river integrate over a greater depth/unit stream bottom area than do those from smaller rivers. These diel NO₃^– patterns are consistent with the influence of photoautotrophic uptake and related denitrification, but we cannot attribute these patterns to assimilation alone because the magnitude and timing of diel dynamics were affected to an unknown extent by processes, such as evapotranspiration, transient storage, and hydrodynamic dispersion. Improvements to diel loss estimates will require additional high-frequency measures, such as dissolved O₂, dissolved organic N, and NH₄⁺, and deployment of 2 measurement stations.