Stream ecosystems receive and transport nutrients from terrestrial ecosystems and are important sites of N retention and removal in catchments. Many streams experience high anthropogenic N loading, which can overwhelm N retention and removal mechanisms and cause large downstream fluxes. Small, headwater streams are important sites of N retention, but the role of streams in larger catchments or as discharge increases is less clear. We evaluated how NO₃⁻ uptake dynamics responded to chronic N loading at different sites in a river draining a large desert catchment (∼7600 km²). Based on nutrient saturation theory, we predicted that chronic N loading would result in decreased uptake efficiency. Previous research suggested that increasing stream discharge also is associated with decreasing N-uptake efficiency. We addressed these relationships for a desert river by examining NO₃⁻ uptake dynamics over variable stream discharge encompassing its long-term range in base flow. We used short-term nutrient-addition studies to estimate uptake parameters for NO₃⁻ in a reference reach and a reach subject to chronic NO₃⁻ input. NO₃⁻ uptake efficiency was lower in the N-enriched reach than in the reference reach. However, within a reach, temporal changes in discharge and N concentration did not always affect uptake efficiency as predicted; e.g., pulses of high N flux following monsoon-season flooding did not result in reduced uptake efficiency. Estimates of denitrification rates indicated that this N-removal process was only a small fraction of N uptake, a result suggesting that most N is temporarily retained and eventually is exported downstream. N concentration exerted the primary influence on NO₃⁻ uptake efficiency in this large desert stream. However, within reaches, other factors that influence N retention, including floods, biota, and variable flow paths, probably contributed to observed temporal variation.