Nitrogen (N) uptake is a key process in stream ecosystems that is mediated mainly by benthic microorganisms (biofilms on different substrata) and has implications for the biogeochemical fluxes at catchment scale and beyond. Here, we focused on the drivers of assimilatory N uptake, especially the effects of hydromorphology and other environmental constraints, across three spatial scales: micro, meso and reach. In two seasons (summer and spring), we performed whole-reach ¹⁵N-labelled ammonium injection experiments in two montane, gravel-bed stream reaches with riffle–pool sequences. N uptake was highest in epilithic biofilms, thallophytes and roots (min–max range 0.2–545.2 mg N m⁻² day⁻¹) and lowest in leaves, wood and fine benthic organic matter (0.05–209.2 mg N m⁻² day⁻¹). At the microscale, N uptake of all primary uptake compartments except wood was higher in riffles than in pools. At the mesoscale, hydromorphology determined the distribution of primary uptake compartments, with fast-flowing riffles being dominated by biologically more active compartments and pools being dominated by biologically less active compartments. Despite a lower biomass of primary uptake compartments, mesoscale N uptake was 1.7–3.0 times higher in riffles than in pools. At reach scale, N uptake ranged from 79.6 to 334.1 mg N m⁻² day⁻¹. Highest reach-scale N uptake was caused by a bloom of thallopyhtes, mainly filamentous autotrophs, during stable low discharge and high light conditions. Our results reveal the important role of hydromorphologic sorting of primary uptake compartments at mesoscale as a controlling factor for reach-scale N uptake in streams.