Models suggest that microbial activity in streams and rivers is a globally significant source of anthropogenic nitrous oxide (N(2)O), a potent greenhouse gas, and the leading cause of stratospheric ozone destruction. However, model estimates of N(2)O emissions are poorly constrained due to a lack of direct measurements of microbial N(2)O production and consequent emissions, particularly from large rivers. We report the first N(2)O budget for a large, nitrogen enriched river, based on direct measurements of N(2)O emissions from the water surface and N(2)O production in the sediments and water column. Maximum N(2)O emissions occurred downstream from Cincinnati, Ohio, a major urban center on the river, due to direct inputs of N(2)O from wastewater treatment plant effluent and higher rates of in situ production. Microbial activity in the water column and sediments was a source of N(2)O, and water column production rates were nearly double those of the sediments. Emissions exhibited strong seasonality with the highest rates observed during the summer and lowest during the winter. Our results indicate N(2)O dynamics in large temperate rivers may be characterized by strong seasonal cycles and production in the pelagic zone.