Nitrogen inputs to the Gulf of Mexico have increased during recent decades and agricultural regions in the upper Midwest, such as those in Illinois, are a major source of N to the Mississippi River. How strongly denitrification affects the transport of nitrate (NO(3)-N) in Illinois streams has not been directly assessed. We used the nutrient spiraling model to assess the role of in-stream denitrification in affecting the concentration and downstream transport of NO(3)-N in five headwater streams in agricultural areas of east-central Illinois. Denitrification in stream sediments was measured approximately monthly from April 2001 through January 2002. Denitrification rates tended to be high (up to 15 mg N m(-2) h(-1)), but the concentration of NO(3)-N in the streams was also high (>7 mg N L(-1)). Uptake velocities for NO(3)-N (uptake rate/concentration) were lower than reported for undisturbed streams, indicating that denitrification was not an efficient N sink relative to the concentration of NO(3)-N in the water column. Denitrification uptake lengths (the average distance NO(3)-N travels before being denitrified) were long and indicated that denitrification in the streambed did not affect the transport of NO(3)-N. Loss rates for NO(3)-N in the streams were <5% d(-1) except during periods of low discharge and low NO(3)-N concentration, which occurred only in late summer and early autumn. Annually, most NO(3)-N in these headwater sites appeared to be exported to downstream water bodies rather than denitrified, suggesting previous estimates of N losses through in-stream denitrification may have been overestimated.