Nitrogen (N) deposition is a chronic input of reactive N to the temperate ecosystems of Appalachian Mountains. Due to the coupled nature of carbon (C) and N cycles, deposition may act as a broad-scale environmental driver of C dynamics if ecosystems are sensitive to excess N. We employed a survey approach along a wide gradient (5 – 32 kg N/ha/yr) of chronic N loading to investigate how N deposition alters C stability by exploring in stream and ground water dissolved organic carbon (DOC) and shifts in: 1) composition, 2) bioavailability, and 3) export from spruce-fir and northern hardwood ecosystems in the Appalachian Mountains. Published experimental plot and survey studies report declines in C storage, DOC:DON, and increased DOC and DON production with N deposition. Thus, we hypothesized that N deposition would increase C bioavailability and C export because N fertilization enhances labile C turnover and stabilizes refractory C reflecting differential responses by microbial enzyme activity responsible for soil C decomposition. Counter to original predictions, we found a strong negative relationship between N deposition and percentage of bioavailable C in stream and groundwater. This pattern was driven by significant increases in the proportion of refractory C in the total DOC pool (243% and 227% for stream and ground water, respectively) for sites experiencing greater than 14 kg N/ha/yr. We contend that N deposition alters carbon stability by organizing DOC bioavailability and export from Appalachian Mountain ecosystems with implications for both C and N cycling in watershed subsystems.