Nitrous oxide (N2O) is an important greenhouse gas in which the main sources are tropical rainforest and agricultural soils. N2O is produced in soils by microbial processes, which are enhanced by the application of nitrogenous fertilizers. The soil N2O bulk isotopic composition (δ
15Nbulk and δ
18O) and the “site-specific,” or intramolecular, 15N isotopic composition, i.e., the 15N/14N ratio at the cenral (α) or terminal (β) nitrogen position, expressed in this study as δ
15N
α
and δ
15N
β
could help identify both the sources (natural and anthropogenic) and microbial pathways of N2O production and consumption prior to emission.We report new isotope measurements of soil N2O emissions and from soil air collected during the rainy season in a mature tropical forest (Tapajos National Forest, Para, Brazil) and in a tropical agricultural corn field (“Fundo Tierra Nueva,” Guárico State, Venezuela). The statistically different δ
15Nbulk emission weighted average between the mature forest (−18.0‰ ± 4.0‰, n = 6) and agricultural corn field (−34.3‰ ± 12.4‰, n = 17) suggest that theδ
15Nbulk data are useful for distinguishing N2O fluxes from fertilized agricultural and natural “background” soils. They also demonstrate that the site-specific δ
15N measurements have the potential to provide a new tool to differentiate between the production and consumption N2O microbiological processes in soils. This study further demonstrates that the observed correlations (or lack thereof) between δ
15N
α
,δ
15N
β
, and δ
18O can be used to estimate the relative proportion of N2O that would have been emitted to the air but was consumed via reduction of N2O to N2 within the soil.