Dissolved organic matter (DOM) dominates the material and energy fluxes within aquatic ecosystems. Carbon fuels the majority of microbial processes, including those that regulate in-stream nitrogen constituents. DOM sources and in situ transformations determine its chemical nature and lability within aquatic systems. Boulder Creek, which is located in the Colorado Front Range and spans an ecosystem gradient from the Continental Divide to the high plains, receives excess atmospheric nitrogen deposition due to its proximity to population centers and agricultural lands. A process-based study was initiated to investigate the relationship between DOM composition, nitrogen cycling, and nitrogen-loads within the watershed. This study was conducted in collaboration with the Niwot Long Term Ecological Research Program and the Boulder Creek Critical Zone Observatory; both historical and recent data document strong organic carbon and inorganic nitrogen gradients that correspond with elevation. Short-term incubations were conducted with freshly collected sediment and water samples to assess denitrification, nitrification, organic nitrogen mineralization, and bulk sediment respiration across these gradients. Denitrification and respiration rates were positively correlated across sites and were negatively related to the humic and fulvic acid content of DOM in the stream sediment. Mineralization rates were also positively correlated with respiration rates. In contrast, there was no cross-site relationship between metabolism and nitrification rates. Nitrification was positively related to the percentage of DOM made up of the large hydrophobic organic acids, presumably due to decreased competition with heterotrophs for available ammonium. We conclude that the quality of available DOM is an important control on the dissimilatory processes regulating the in-stream nitrogen cycle within Boulder Creek and provide insight as to how the watershed will respond to increased atmospheric nitrogen deposition.