Streams that drain agricultural watersheds in the midwestern US deliver large N loads to downstream water bodies. Denitrification is a potential sink for N in streams, but its importance in agricultural streams is unclear. Denitrification was examined in Big Ditch, a NO₃-rich tributary to the Sangamon River in east-central Illinois. Denitrification associated with benthic sediments and floating mats of algae and macrophytes was measured from May to November 2002. Daily NO₃-N loads were calculated for the 2002 calendar year to provide a context for the denitrification rates. Four other streams were sampled less intensively and the results indicated that Big Ditch was typical of agricultural streams in the region. During the growing season, plant biomass in Big Ditch was occasionally >200 g dry mass (DM)/m², but biomass declined sharply following scouring spates in June and August. Maximum rates of plant-associated and sediment denitrification were similar on a DM basis (4.2 and 3.7 μg N₂O [g DM]⁻¹ h⁻¹, respectively). However, denitrification rates in the sediments were more than an order of magnitude greater than the rates associated with plant material on an areal basis. Large floating mats of algae and macrophytes often covered much of Big Ditch, but were not major sites for denitrification. Denitrification rates in the sediments generally were higher than those reported from other streams, with a maximum value of 15.8 mg N m⁻² h⁻¹, but daily NO₃-N loads in Big Ditch often were >5 Mg N/d (Mg = 10⁶ g). Denitrification rates were high at times, but instream denitrification appeared not to substantially affect N losses from this agricultural watershed.