Nitrous oxide (N(2)O) is a potent greenhouse gas that contributes to climate change and stratospheric ozone destruction. Anthropogenic nitrogen (N) loading to river networks is a potentially important source of N(2)O via microbial denitrification that converts N to N(2)O and dinitrogen (N(2)). The fraction of denitrified N that escapes as N(2)O rather than N(2) (i.e., the N(2)O yield) is an important determinant of how much N(2)O is produced by river networks, but little is known about the N(2)O yield in flowing waters. Here, we present the results of whole-stream (15)N-tracer additions conducted in 72 headwater streams draining multiple land-use types across the United States. We found that stream denitrification produces N(2)O at rates that increase with stream water nitrate (NO(3)(-)) concentrations, but that <1% of denitrified N is converted to N(2)O. Unlike some previous studies, we found no relationship between the N(2)O yield and stream water NO(3)(-). We suggest that increased stream NO(3)(-) loading stimulates denitrification and concomitant N(2)O production, but does not increase the N(2)O yield. In our study, most streams were sources of N(2)O to the atmosphere and the highest emission rates were observed in streams draining urban basins. Using a global river network model, we estimate that microbial N transformations (e.g., denitrification and nitrification) convert at least 0.68 Tg·y(-1) of anthropogenic N inputs to N(2)O in river networks, equivalent to 10% of the global anthropogenic N(2)O emission rate. This estimate of stream and river N(2)O emissions is three times greater than estimated by the Intergovernmental Panel on Climate Change.