In-stream nitrogen processing in the Mississippi River has been suggested as one mechanism to reduce coastal eutrophication in the Gulf of Mexico. Aquatic macrophytes in river channels and flood plain lakes have the potential to temporarily remove large quantities of nitrogen through assimilation both by themselves and by the attached epiphyton. In addition, rooted macrophytes act as oxygen pumps, creating aerobic microsites around their roots where coupled nitrification–denitrification can occur. We used in situ ¹⁵N–NO₃ ⁻ tracer mesocosm experiments to measure nitrate assimilation rates for macrophytes, epiphyton, and microbial fauna in the sediment in Third Lake, a backwater lake of the upper Mississippi River during June and July 2005. We measured assimilation over a range of nitrate concentrations and estimated a nitrate mass balance for Third Lake. Macrophytes assimilated the most nitrate (29.5 mg N m⁻² d⁻¹) followed by sediment microbes (14.4 mg N m⁻² d⁻¹) and epiphytes (5.7 mg N m⁻² d⁻¹)_. Assimilation accounted for 6.8% in June and 18.6% in July of total nitrate loss in the control chambers. However, denitrification (292.4 mg N m⁻² d⁻¹) is estimated to account for the majority (82%) of the nitrate loss. Assimilation and denitrification rates generally increased with increasing nitrate concentration but denitrification rates plateaued at about 5 mg N L⁻¹. This suggests that backwaters have the potential to remove a relatively high amount of nitrate but will likely become saturated if the load becomes too large.