We evaluated patterns of denitrification and factors effecting denitrification in the upper Mississippi River. Measurements were taken over 2 years, during which river discharge ranged from record flooding to base flow conditions. Over the period of study, average denitrification enzyme activity was highest in backwater lakes and lowest in the main channel. Throughout the study reach, highest denitrification enzyme activity occurred during fall and lowest occurred in winter. Rates during spring floods (2001) were only slightly higher than during the preceding winter. Mean unamended denitrification rates ranged from 0.02 (fall 2001 in backwaters) to 0.40 ??g N??cm -2??h-1 (spring 2001 in backwaters). Laboratory experiments showed that denitrification rates increased significantly with addition of NO3- regardless of sediment C content, while rates increased little with addition of labile C (glucose). Denitrification in this reach of the upper Mississippi River appears to be NO3- limited throughout the growing season and the delivery of NO 3- is strongly controlled by river discharge and hydrologie connectivity across the floodplain. We estimate that denitrification removes 6939 t N??year-1 or 6.9% of the total annual NO 3- input to the reach. Hydrologic connectivity and resultant NO3- delivery to high-C sediments is a critical determinant of reach-scale processing of N in this floodplain system.