We report measurements of the ¹⁵N and ¹⁸O signature of N₂O emitted from tropical rain forest soils at the La Selva Biological station in Costa Rica and in the Fazenda Vitoria in the State of Pará, Brazil. The δ¹⁵N values ranged from −34 to 2‰ with respect to atmospheric N₂, while δ¹⁸O values had a smaller range, from −4 to 18‰ with respect to atmospheric O₂. We attribute these large variations to differences in microbial production, consumption, and transport of N₂O. In general the δ¹⁵N of N₂O emissions from an Oxisol soil in Brazil were consistently enriched by ∼20‰ in ¹⁵N compared to those from Ultisol and Inceptisol soils in Costa Rica. Denitrification is the most likely source of N₂O in both locations during the rainy season, and the ¹⁵N of nitrate was similar in both locations. We attribute the overall variability in emitted ¹⁵N to differences in the ratio of N₂O:N₂ escaping from the soil to the atmosphere, with a larger fraction of the N₂O reduced to N₂ at the Brazilian sites. We found light δ¹⁵N-N₂O values associated with high N₂O emissions in a fertilized agricultural site in Costa Rica and in a “hot spot” of high emissions in the forest site in Brazil. This result suggests that the increase of substrate availability might increase the fractionation associated with N₂O production. Overall, the Brazilian Oxisol soils had the most enriched δ¹⁵N-N₂O emissions yet measured from soils. If these are more representative of tropical soil emissions than the Costa Rica emissions, then the globally averaged δ¹⁵N-N₂O tropical rain forest soil source is more enriched than previously estimated. The large variations in isotopic signature for N₂O emissions demonstrate the potential utility of stable isotopes as tools for understanding the processes of N₂O production and consumption in soils.