The LINX II (Lotic Intersite Nitrogen eXperiment) project was designed to quantify the rates and mechanisms of nitrate retention in streams using stable isotope tracer additions. The study encompassed 72 stream reaches spread across 8 North American biomes. Within each biome, 9 streams were selected in three watershed land-use categories: 3 reference, 3 agricultural, and 3 urbanized. The core of the study was a 24-hour release of 15N- labeled nitrate. Prior to the isotope addition, physical, chemical and biological characteristics of the stream were measured. The measurements included, but were not limited to, dissolved nutrient concentrations, dissolved conservative tracer additions (to quantify hydraulic and hyporheic retention, velocity and discharge), standing stocks of primary uptake biota (including suspended and benthic particulate materials) as well as channel dimensions, photosynthetically active radiation, and water temperature. During the isotope release, whole stream rates of ecosystem metabolism were quantified (including quantification of re-aeration coefficients using tracer gas additions), and concentrations of 15N-labeled NO3, NH4, N2 and N2O were measured. Immediately following the isotope addition, 15N uptake by aquatic organisms was quantified by sampling biomass components on the stream bed. The data generated from these 72 stream reaches were used to develop a stream nitrogen retention model for each biome, which was expanded to entire drainage networks to predict nitrogen fluxes. The LINX II study demonstrated how biotic uptake of nitrate and denitrification increased with increasing nitrate concentrations. However, the efficiency of total uptake and denitrification actually declined with increasing nitrate concentrations (such as those seen on agricultural or urbanized streams), yielding higher rates of dissolved nitrogen exports downstream. The datasets presented here consist of the primary data collected by the LINX II study participants.