The first step of autotrophic nitrification is performed by ammonia-oxidizing archaea (AOA) and bacteria (AOB). Recent studies show that their relative contributions are determined by the substrate sources and availability, yet evidence provided by quantification of their respective gross activities in soil is lacking. Here, we conducted a microcosm study with agricultural soil with high (50 μg N g−1) and low (5 μg N g−1) ammonium application, and quantified gross nitrification rates using 15N-tracers. AOA and AOB activities were distinguished using the bacterial inhibitor 1-octyne and acetylene, which inhibits both AOA and AOB. Under low ammonium supply, AOA and AOB contributed equally to gross ammonia oxidation, but AOB outcompeted AOA under higher ammonium supply. These results provide the first direct evidence that substrate availability affects the relative contribution of AOA and AOB to gross nitrification. Display omitted •First quantification of specific gross nitrification rates for AOA and AOB.•AOB outcompeted AOA at high NH4+ application.•Importance of AOA for gross nitrification was high for soils with low NH4+ supply.