Ammonia-oxidizing microbes control the rate-limiting step of nitrification, a critical ecosystem process, which affects retention and mobility of nitrogen in soil ecosystems. This study investigated substrate (NH₄+) and nutrient (K and P) limitation of ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) in temperate forest soils at Coweeta Hydrologic Laboratory, a long-term ecological research site in western North Carolina, USA. We investigated substrate and nutrient limitation by amending soils with either ammonium or a nutrient solution containing P and K, then assessing the growth of these organisms during in situ soil incubations. We found substantial growth of both AOA and AOB during all incubations including unamended control incubations. Our results demonstrate that substrate availability limits nitrification by AOB and that high levels of substrate addition inhibit the growth of AOA in these soils. We found no evidence for nutrient limitation of AOB, though nutrient addition indirectly stimulated nitrification by AOB through increased nitrogen mineralization. Our data did suggest nutrient limitation by AOA, though it is unclear whether AOA significantly contribute to ammonia oxidation in this system. Furthermore, we show that AOB are responsible for the majority of ammonia oxidation in high substrate, high nutrient conditions.