High latitude, boreal watersheds are nitrogen (N)-limited ecosystems that export large amounts of organic carbon (C). Key controls on C cycling in these environments are the biogeochemical processes affecting the N cycle. A study was conducted in Nome Creek, an upland headwater tributary of the Yukon River, and two first-order tributaries to Nome Creek, to examine the relation between seasonal and transport-associated changes in C and N pools and N-cycling processes across varying hydrologic gradients using laboratory bioassays of water and sediment samples and in-stream tracer tests. DON exceeded dissolved inorganic nitrogen (DIN) in Nome Creek except late in the summer season, with little variation in organic C:N ratios with time or transport distance. DIN was dominant in the 1st order tributaries. Rates of organic N mineralization and denitrification in laboratory incubations were related to sediment organic C content, while nitrification rates differed greatly between two 1st order tributaries with similar drainages. Additions of DIN or urea did not stimulate microbial activity. In-stream tracer tests with nitrate and urea indicated that uptake rates were slow relative to transport rates; simulated rates of uptake in stream storage zones were higher than rates assessed in the laboratory bioassays. In general, N-cycle processes were more active and had a greater overall impact in the 1st order tributaries and were minimized in Nome Creek, the larger, higher velocity, transport-dominated stream. Understanding key controls on N-cycling processes in these watersheds has important implications for DIN speciation and down-stream impacts of potential increased N loads in response to climate warming.