Anthropogenic nitrate (NO3-) within the Mississippi-Atchafalaya River basin and discharge to the Gulf of Mexico has been linked to serious environmental problems. The sources of this NO 3- have been estimated by others using mass balance methods; however, there is considerable uncertainty in these estimates. Part of the uncertainty is the degree of denitrification that the NO3- has undergone. The isotopic composition of NO3- in the Mississippi River adjacent to Illinois and tile drain (subsurface drain) discharge in agricultural areas of east-central Illinois was examined using N and O isotopes to help identify the major sources of NO 3- and assess the degree of denitrification in the samples. The isotopic evidence suggests that most of the NO3- in the river is primarily derived from synthetic fertilizers and soil organic N, which is consistent with published estimates of N inputs to the Mississippi River. The 1:2 relationship between ??18O and ??15N also indicate that, depending on sample location and season, NO3- in the river and tile drains lias undergone significant denitrification, ranging from about 0 to 55%. The majority of the denitrification appears to have occurred before discharge into the Mississippi River. ?? ASA, CSSA, SSSA.