We conducted a two-year investigation into the sources and fate of nitrate (NO3-) in the Illinois River from the Chicago area to the river's confluence with the Mississippi River. Samples from waterways in the Chicago area (Des Plaines River and the Sanitary and Ship Canal) had relatively high concentrations of nitrogen (N) species and NO3- isotopic compositions indicative of treated wastewater (TWW). Downstream of the Brandon Road Lock and Dam, NO3- in tributaries discharging to the Illinois River primarily comes from tiles draining row crops. Nitrate isotopic signatures from these tributaries as well as drain tiles were indicative of synthetic fertilizer and/or soil organic matter (SOM) at various stages of denitrification. Nitrate-N concentrations generally decreased in the Illinois River with distance from the Chicago area primarily due to dilution. The decrease in NO3-N concentrations was especially conspicuous during the summer, when there is minimal discharge from drain tiles and NO3-N concentrations in the tributaries were low. In August 2005, when conditions were very dry, NO3-N concentrations decreased from 7.4 mg/L in the Chicago area to less than 1 mg/L near where the Illinois River discharges to the Mississippi River. The isotopic composition of NO3- in water samples from the Illinois River were a mixture of three end members: (1) fertilizer and/or SOM in drain tile water, typically showing the least amount of denitrification, (2) fertilizer and/or SOM in deeper ground water, showing the highest degree of denitrification, and (3) TWW. There was seasonal variability, depending on the volume of water flowing in the Illinois River. During high flow periods, river water samples plotted closest to those of tile drain samples; during low flow periods, a greater influence of TWW was observed in the isotopic composition. A subset of summer samples from the Chicago waterways had isotopic values plotting near and within the domain that characterizes manure and sewage. Nitrate in the Chicago area is primarily derived from TWW, with its isotopic signature evident downstream at least as far as Pekin during most of the year and all the way to the Mississippi River during periods of low flow. Denitrification occurs predominantly in groundwater between and away from drain tiles, although there is evidence that in-stream denitrification and/or biological uptake of NO3- occurs in the Peoria Lake reach of the Illinois River, at least during periods of low flow in the summer. We calculated that the river was losing about half of its NO3-N load in Peoria Lake in August 2005 (a period of very low flow), at a rate of about 7500 kg/day.