We investigated variations in resource availability (nitrate and labile organic carbon, LOC) as determinants of denitrification in sediments of streams in the southern Appalachian Mountains, USA. Stream water and sediments were sampled seasonally in two streams of contrasting nitrate availability, Noland Creek (high NO₃-N) and Walker Branch (low NO3-N). Eight additional streams with varying nitrate levels were sampled once during summer. Stream sediments were incubated at ambient stream temperatures, and nitrous oxide accumulation was quantified following acetylene inhibition of nitrous oxide reduction. Denitrification potential was greater in Noland Creek than Walker Branch and was generally greater in sediments from the higher-nitrate streams. In autumn and spring, nitrate and LOC amendments indicated that denitrification potential in Walker Branch sediments was nitrate limited, with temperature having no effect on rates. Denitrification potential in Noland Creek sediments was not limited by nitrate, but temperature had a significant effect. When Noland Creek seasonal data were corrected to a common temperature, no seasonal differences in denitrification potential were detected. Nitrate-N in the 10 surveyed streams ranged from 10 to 549 mg/L, with the highest NO₃-N levels and denitrification rates generally occurring in the higher elevation streams in the GSMNP. We found that nitrate availability, more than LOC availability, controls potential denitrification in these streams.