Agricultural drainage can contribute excess N to aquatic ecosystems. The objective of our study was to investigate the capacity of agricultural ditches to remove NO₃⁻ via denitrification in maintained 1-stage and naturalized 2-stage agricultural ditches. We hypothesized that maintenance of ditches limits the potential for denitrification by removing in-stream and riparian vegetation and excavating fine sediments. We quantified denitrification rates in the sediments collected from ten 1-stage and ten 2-stage headwater ditches with 2 methods (sediment static core and denitrification enzyme activity) and over 4 sampling periods. We also measured water, plant, and sediment characteristics. With both methods, denitrification rates from sediments collected inside the channel (i.e., not along the slope and the bench) did not differ between the 2 types of ditch. We used a series of enrichment treatments of sediment slurries with C and NO₃⁻ to determine that denitrification was limited by NO₃⁻ on the bench of the 2-stage ditches and by both NO₃⁻ and C on the slope of the 1-stage ditches. We hypothesize that greater denitrification rates measured with the static core method in the slope might have been linked to greater % fine sediments in the slope of the 1-stage ditches and, thus, more developed anaerobic conditions in the sediment cores. Accumulation of organic matter in the benches that form in unmaintained ditches is favorable to denitrification, as shown by greater denitrification measured in the sediment slurries unamended with C.