A study was conducted to determine whether NO3 stable isotopes (δ15N and δ18O), at natural abundance levels, could discriminate among NO3 sources from sites with different land uses at the basin scale. Water samples were collected from 24 sites in the Mississippi River Basin from five land-use categories: (1) large river basins (>34 590 km2) draining multiple land uses and smaller basins in which the predominant land use was (2) urban, (3) undeveloped, (4) crops, or (5) crops and livestock. Our data suggest that riverine nitrates from different land uses have overlapping but moderately distinct isotopic signatures. δ18O data were critical in showing abrupt changes in NO3 source with discharge. The isotopic values of large rivers resembled crop sites, sites with livestock tended to have δ15N values characteristic of manure, and urban sites tended to have high δ18O values characteristic of atmospheric nitrate.