Denitrification is an important net sink for NO₃⁻ in streams, but direct measurements are limited and in situ controlling factors are not well known. We measured denitrification at multiple scales over a range of flow conditions and NO₃⁻ concentrations in streams draining agricultural land in the upper Mississippi River basin. Comparisons of reach-scale measurements (in-stream mass transport and tracer tests) with local-scale in situ measurements (pore-water profiles, benthic chambers) and laboratory data (sediment core microcosms) gave evidence for heterogeneity in factors affecting benthic denitrification both temporally (e.g., seasonal variation in NO₃⁻ concentrations and loads, flood-related disruption and re-growth of benthic communities and organic deposits) and spatially (e.g., local stream morphology and sediment characteristics). When expressed as vertical denitrification flux per unit area of streambed (U_denit, in μmol N m⁻² h⁻¹), results of different methods for a given set of conditions commonly were in agreement within a factor of 2–3. At approximately constant temperature (~20 ± 4°C) and with minimal benthic disturbance, our aggregated data indicated an overall positive relation between U_denit (~0–4,000 μmol N m⁻² h⁻¹) and stream NO₃⁻concentration (~20–1,100 μmol L⁻¹) representing seasonal variation from spring high flow (high NO₃⁻) to late summer low flow (low NO₃⁻). The temporal dependence of U_denit on NO₃⁻was less than first-order and could be described about equally well with power-law or saturation equations (e.g., for the unweighted dataset, U_denit ≈26 * [NO₃⁻]^0.44 or U_denit≈640 * [NO₃⁻]/[180 + NO₃⁻]; for a partially weighted dataset, U_denit ≈14 * [NO₃⁻]^0.54 or U_denit ≈700 * [NO₃⁻]/[320 + NO₃⁻]). Similar parameters were derived from a recent spatial comparison of stream denitrification extending to lower NO₃⁻ concentrations (LINX2), and from the combined dataset from both studies over 3 orders of magnitude in NO₃⁻concentration. Hypothetical models based on our results illustrate: (1) U_denit was inversely related to denitrification rate constant (k1_denit, in day⁻¹) and vertical transfer velocity (v_f,denit, in m day⁻¹) at seasonal and possibly event time scales; (2) although k1_denit was relatively large at low flow (low NO₃⁻), its impact on annual loads was relatively small because higher concentrations and loads at high flow were not fully compensated by increases in U_denit; and (3) although NO₃⁻ assimilation and denitrification were linked through production of organic reactants, rates of NO₃⁻ loss by these processes may have been partially decoupled by changes in flow and sediment transport. Whereas k1_denit and v_f,denit are linked implicitly with stream depth, NO₃⁻ concentration, and(or) NO₃⁻ load, estimates of U_denit may be related more directly to field factors (including NO₃⁻ concentration) affecting denitrification rates in benthic sediments. Regional regressions and simulations of benthic denitrification in stream networks might be improved by including a non-linear relation between U_denit and stream NO₃⁻concentration and accounting for temporal variation.