AbstractPasture-based livestock systems are often associated with losses of reactive forms of nitrogen (N) to the environment. Research has focused on losses to air and water due to the health, economic and environmental impacts of reactive N. Di-nitrogen (N₂) emissions are still poorly characterized, both in terms of the processes involved and their magnitude, due to financial and methodological constraints. Relatively few studies have focused on quantifying N₂ losses in vivo and fewer still have examined the relative contribution of the different N₂ emission processes, particularly in grazed pastures. We used a combination of a high ¹⁵N isotopic enrichment of applied N with a high precision of determination of ¹⁵N isotopic enrichment by isotope-ratio mass spectrometry to measure N₂ emissions in the field. We report that 55.8 g N m⁻² (95%, CI 38 to 77 g m⁻²) was emitted as N₂ by the process of co-denitrification in pastoral soils over 123 days following urine deposition (100 g N m⁻²), compared to only 1.1 g N m⁻² (0.4 to 2.8 g m⁻²) from denitrification. This study provides strong evidence for co-denitrification as a major N₂ production pathway, which has significant implications for understanding the N budgets of pastoral ecosystems.