Nitrate (NO3) and other nutrients discharged by the Mississippi River are suspected of causing a zone of depleted dissolved oxygen (hypoxic zone) in the Gulf of Mexico each summer. The hypoxic zone may have an adverse affect on aquatic life and commercial fisheries. The amount of NO3 delivered by the Mississippi River to the Gulf of Mexico is well documented, but the relative contributions of different sources of NO3, and the magnitude of subsequent in-stream transformations of NO3, are not well understood. Forty-two water samples collected in 1997 and 1998 at eight stations located either on the Mississippi River or its major tributaries were analysed for NO3, total nitrogen (N), atrazine, chloride concentrations and NO3 stable isotopes (δ15N and δ18O). These data are used to assess the magnitude and nature of in-stream N transformation and to determine if the δ15N and δ18O of NO3 provide information about NO3 sources and transformation processes in a large river system (drainage area 2 900 000 km2) that would otherwise be unavailable using concentration and discharge data alone. Results from 42 samples indicate that the δ15N and δ18O ratios between sites on the Mississippi River and its tributaries are somewhat distinctive, and vary with season and discharge rate. Of particular interest are two nearly Lagrangian sample sets, in which samples from the Mississippi River at St Francisville, LA, are compared with samples collected from the Ohio River at Grand Chain, II, and the Mississippi River at Thebes, IL. In both Lagrangian sets, mass-balance calculations indicate only a small amount of in-stream N loss. The stable isotope data from the samples suggest that in-stream N assimilation and not denitrification accounts for most of the N loss in the lower Mississippi River during the spring and early summer months.