Nitrogen budgets established for large river systems reveal that up to 60% of the nitrate exported from agricultural soils is eliminated, either when crossing riparian wetlands areas before even reaching surface waters, or within the rivers themselves through benthic denitrification. The study of isotopic composition of riverine nitrates could offer an elegant means to asses the extent of denitrification and thus confirm these budgets, as it is known that denitrification results in a natural 15N and 18O enrichment of residual nitrates. The results reported here, for the Seine river system (France), demonstrate the feasibility of this isotopic approach at the scale of large watersheds. We first show, based on in situ observations carried out in a large storage reservoir in the upstream Seine catchment (Der lake), where an intensive benthic denitrification occurs, as well as based on laboratory experiments of denitrification under controlled conditions, that the isotopic discrimination associated to benthic denitrification is minimal probably because the rate limiting step of the process consists of nitrate diffusion through the water-sediment interface. Riparian denitrification on the contrary, when it implies nitrate reduction during convective transfer through reducing environments, causes a much more significant isotopic enrichment of residual nitrate. We report measurements of nitrogen isotopic composition of nitrate from rivers of various stream orders in the Seine river system, under summer conditions. Anomalies exist in the data with respect to the values expected from the mixture of the various sources of nitrate: we attribute these to riparian denitrification. We show however that, because of the non uniform distribution and activity of denitrifying riparian zones along tributaries, the isotopic signature they confer to residual nitrate in river water intrinsically provides only a minimum estimate of the extent of denitrification.