Rates of benthic denitrification were measured using two techniques, membrane inlet mass spectrometry (MIMS) and isotope ratio mass spectrometry (IRMS), applied to sediment cores from two NO3−-rich streams draining agricultural land in the upper Mississippi River Basin. Denitrification was estimated simultaneously from measurements of N2:Ar (MIMS) and 15N[N2] (IRMS) after the addition of low-level 15NO3− tracer (15N:N = 0.03–0.08) in stream water overlying intact sediment cores. Denitrification rates ranged from about 0 to 4400 μmol N·m−2·h−1 in Sugar Creek and from 0 to 1300 μmol N·m−2·h−1 in Iroquois River, the latter of which possesses greater streamflow discharge and a more homogeneous streambed and water column. Within the uncertainties of the two techniques, there is good agreement between the MIMS and IRMS results, which indicates that the production of N2 by the coupled process of nitrification/denitrification was relatively unimportant and surface-water NO3− was the dominant source of NO3− for benthic denitrification in these streams. Variation in stream NO3− concentration (from about 20 μmol/L during low discharge to 1000 μmol/L during high discharge) was a significant control of benthic denitrification rates, judging from the more abundant MIMS data. The interpretation that NO3− concentration directly affects denitrification rate was corroborated by increased rates of denitrification in cores amended with NO3−. Denitrification in Sugar Creek removed ≤11% per day of the in-stream NO3− in late spring and removed roughly 15–20% in late summer. The fraction of NO3− removed in Iroquois River was less than that of Sugar Creek. Although benthic denitrification rates were relatively high during periods of high stream flow, when NO3 concentrations were also high, the increase in benthic denitrification could not compensate for the much larger increase in stream NO3− fluxes during high flow. Consequently, fractional NO3− losses were relatively low during high flow.