Studies have repeatedly shown that agricultural and urban areas export considerably more nitrogen to streams than forested counterparts, yet it is difficult to identify and quantify nitrogen sources to streams due to complications associated with terrestrial and in-stream biogeochemical processes. In this study, we used the isotopic composition of nitrate (delta15N-NO3- and delta18O-NO3-) in conjunction with a simple numerical model to examine the spatial and temporal variability of nitrate (NO3-) export across a land-use gradient and how agricultural and urban development affects net removal mechanisms. In an effort to isolate the effects of land use, we chose small headwater systems in close proximity to each other, limiting the variation in geology, surficial materials, and climate between sites. The delta15N and delta18O of stream NO3- varied significantly between urban, agricultural, and forested watersheds, indicating that nitrogen sources are the primary determinant of the delta15N-NO3-, while the delta18O-NO3- was found to reflect biogeochemical processes. The greatest NO3- concentrations corresponded with the highest stream delta15N-NO3- values due to the enriched nature of two dominant anthropogenic sources, septic and manure, within the urban and agricultural watersheds, respectively. On average, net removal of the available NO3- pool within urban and agricultural catchments was estimated at 45%. The variation in the estimated net removal of NO3- from developed watersheds was related to both drainage area and the availability of organic carbon. The determination of differentiated isotopic land-use signatures and dominant seasonal mechanisms illustrates the usefulness of this approach in examining the sources and processing of excess nitrogen within headwater catchments.