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Abstract Nitrogen loss from cultivated soils threatens the economic and environmental sustainability of agriculture. Nitrate (NO 3 − ) derived from nitrification of nitrogen fertilizer and ammonified soil organic nitrogen may be lost from soils via denitrification, producing dinitrogen gas (N 2 ) or the greenhouse gas nitrous oxide (N 2 O). Nitrate that accumulates in soils is also subject to leaching loss, which can degrade water quality and make NO 3 − available for downstream denitrification. Here we use patterns in the isotopic composition of NO 3 − observed from 2012 to 2017 to characterize N loss to denitrification within soils, groundwater, and stream riparian corridors of a non-irrigated agroecosystem in the northern Great Plains (Judith River Watershed, Montana, USA). We find evidence for denitrification across these domains, expressed as a positive linear relationship between δ 15 N and δ 18 O values of NO 3 − , as well as increasing δ 15 N values with decreasing NO 3 − concentration. In soils, isotopic evidence of denitrification was present during fallow periods (no crop growing), despite net accumulation of NO 3 − from the nitrification of ammonified soil organic nitrogen. We combine previous results for soil NO 3more »− mass balance with δ 15 N mass balance to estimate denitrification rates in soil relative to groundwater and streams. Substantial denitrification from soils during fallow periods may be masked by nitrification of ammonified soil organic nitrogen, representing a hidden loss of soil organic nitrogen and an under-quantified flux of N to the atmosphere. Globally, cultivated land spends ca. 50% of time in a fallow condition; denitrification in fallow soils may be an overlooked but globally significant source of agricultural N 2 O emissions, which must be reduced along-side other emissions to meet Paris Agreement goals for slowing global temperature increase.« less