Sediments in the backwaters of the Upper Mississippi River (UMR) are highly organic and provide an optimal environment for N removal. We monitored an 8.6-ha UMR backwater site near La Crosse, Wisconsin, for nearly 3 y to assess temporal variability, seasonal trends, and the factors regulating denitrification. We measured rates of unamended denitrification (DEN) and denitrification enzyme activity (DEA) rates at ambient temperature and DEA at 30 degrees C (DEA30). Seasonal mean (+/- 1 SE) DEN rates ranged from 0.041 +/- 0.015 to 0.47 +/- 0.23 mu g N cm(-2) h(-1)and were highest in winter and lowest in autumn. Seasonal rates of DEA exhibited a different pattern with the highest rates in summer (25.6 +/- 3.4 mu g N cm(-2) h(-1)) and the lowest rates in winter (10.6 +/- 2.1 mu g N cm(-2) h(-1)). The overall mean DEA30 rate was 31.0 +/- 1.9 mu g N cm(-2) h(-1) but showed no significant seasonal pattern. Short-term (weekly) and seasonal variability exhibited by rates of DEN and DEA were best explained by water-column NO3- concentration and temperature, respectively. No environmental variables explained a significant amount of variability in DEA30. Our results suggest that nutrient (i.e., NO3-) availability and temperature are both regulators of denitrification, with NO3- concentration being the most important limiting factor in this system. The high DEN rates during winter were in response to elevated NO3- concentrations resulting from a chain reaction beginning with algal blooms creating oxic conditions that stimulated nitrification. Increasing hydrological connectivity in large rivers as a river management tool to reduce N flux to downstream areas may be beneficial.