The isotopic signatures of ¹⁵N and ¹⁸O in N₂O emitted from tropical soils vary both spatially and temporally, leading to large uncertainty in the overall tropical source signature and thereby limiting the utility of isotopes in constraining the global N₂O budget. Determining the reasons for spatial and temporal variations in isotope signatures requires that we know the isotope enrichment factors for nitrification and denitrification, the two processes that produce N₂O in soils. We have devised a method for measuring these enrichment factors using soil incubation experiments and report results from this method for three rain forest soils collected in the Brazilian Amazon: soil with differing sand and clay content from the Tapajos National Forest (TNF) near Santarém, Pará, and Nova Vida Farm, Rondônia. The ¹⁵N enrichment factors for nitrification and denitrification differ with soil texture and site: −111‰ ± 12‰ and −31‰ ± 11‰ for a clay-rich Oxisol (TNF), −102‰ ± 5‰ and −45‰ ± 5‰ for a sandier Ultisol (TNF), and −10.4‰ ± 3.5‰ (enrichment factor for denitrification) for another Ultisol (Nova Vida) soil, respectively. We also show that the isotopomer site preference (δ¹⁵N^α − δ¹⁵N^β _, where α indicates the central nitrogen atom and β the terminal nitrogen atom in N₂O) may allow differentiation between processes of production and consumption of N₂O and can potentially be used to determine the contributions of nitrification and denitrification. The site preferences for nitrification and denitrification from the TNF-Ultisol incubated soils are: 4.2‰ ± 8.4‰ and 31.6‰ ± 8.1‰, respectively. Thus, nitrifying and denitrifying bacteria populations under the conditions of our study exhibit significantly different ¹⁵N site preference fingerprints. Our data set strongly suggests that N₂O isotopomers can be used in concert with traditional N₂O stable isotope measurements as constraints to differentiate microbial N₂O processes in soil and will contribute to interpretations of the isotopic site preference N₂O values found in the free troposphere.