Implementation of the Clean Water Act might be further refined to recognize differences in biological availability of different nitrogen species—nitrate-N, ammonium-N, and dissolved organic nitrogen (DON)—in the regulation of total nitrogen (TN) total maximum daily loads (TMDL). This study was conducted to assess the location and importance of N loading and associated nutrient limitation in the Truckee River across the urban gradient. To refine understanding of nutrient loading effects, periphyton nutrient limitation assays were performed across the urban gradient. Since the variety of constituents in DON have not been thoroughly identified and may vary, based on source and season, 67 day bioassays were performed to measure the fraction of DON that is mineralized or converted to particulate matter. Specifically, the bioavailable fraction of wastewater and urban DON was compared to “naturally derived” DON in the receiving river system. Seasonal bioassays in 2007 and 2008 demonstrated that wastewater derived DON consistently had a higher bioavailable fraction (20-51%) than naturally derived DON, which was usually a small fraction. However during summer 2007 the fraction of bioavailable DON was similar for wastewater and natural sources (40% and 41%, respectively). DON derived from urban runoff had the highest degree of variation in bioavailability (3-70%) as opposed to the more consistent bioavailability of wastewater DON or the summer pulse of bioavailblity in naturally derived DON. In the river, downstream from wastewater discharge, the bioavailable fraction of DON varied seasonally (0-42%). Periphyton nutrient limitation assays demonstrated variations in significant limitation (p<0.05) between seasons and sites: (1) upstream of the urban gradient during the summer 2007 (N) and spring-fall 2008 (N+P), (2) within the urban gradient during the spring-fall 2008 (N,2° P), (3) in the urban outfall during Fall 2008 (N), and (4) downstream during spring 2008 (N+P). A significant increase in baseline (control) periphyton growth across the urban gradient was observed for spring, summer, and fall 2008 (p<0.05). It appears that wastewater DON consistently has a significant bioavailable fraction (20-51%) and a recalcitrant fraction (49-80%) which suggests TMDLs could be altered to regulate the bioavailable fraction of TN. In-stream DON bioavailability varies seasonally and across the urban gradient. The shift from co-limitation (N+P) to N limitation (2° P) to no limitation and increases in baseline periphyton production across the urban gradient during several seasons suggests that appropriate N TMDLs are important and that urban nutrient loads significantly affect this river system.