AbstractConsidering recent increases in anthropogenic N loading, it is essential to identify the controls on N removal and retention in aquatic ecosystems because the fate of N has consequences for water quality in streams and downstream ecosystems. Biological uptake of nitrate (NO3−) is a major pathway by which N is removed from these ecosystems. Here we used data from the second Lotic Intersite Nitrogen eXperiment (LINX II) in a multivariate analysis to identify the primary drivers of variation in NO3− uptake velocity among biomes. Across 69 study watersheds in North America, dissolved organic carbon:NO3− ratios and photosynthetically active radiation were identified as the two most important predictor variables in explaining NO3− uptake velocity. However, within a specific biome the predictor variables of NO3− uptake velocity varied and included various physical, chemical, and biological attributes. Our analysis demonstrates the broad control of elemental stoichiometry on NO3− uptake velocity as well as the importance of biome‐specific predictors. Understanding this spatial variation has important implications for biome‐specific watershed management and the downstream export of NO3−, as well as for development of spatially explicit global models that describe N dynamics in streams and rivers.