Significance Reactive nitrogen oxides (NO _y ) can negatively impact air quality, visibility, and human health. Biogenic sources of NO _y are poorly understood despite their growing importance in future scenarios of decreasing anthropogenic emissions. Using soil from two forest stands that differ in plant and soil characteristics, we show that ammonia-oxidizing bacteria and to a lesser extent, heterotrophic microbes are the primary producers of NO _y . Moreover, we show that ammonia-oxidizing archaea contribute little to NO _y yet play a central role in determining nitrogen-cycle rates in forests. Given known relationships between tree species and soil characteristics, we conclude that NO _y emissions are spatially variable, occurring in hotspots throughout the eastern United States.