Isotopic signatures can be used to study sink and source processes of N2O, but the success of this approach is limited by insufficient knowledge on the isotope fractionation factors of the various reaction pathways. We investigated isotope enrichment factors of the N2O-to-N2 step of denitrification (ε) in two arable soils, a silt-loam Haplic Luvisol and a sandy Gleyic Podzol. In addition to the ε of 18O (ε18O) and of average 15N (εbulk), the ε of the 15N site preference within the linear N2O molecule (εSP) was also determined. Soils were anaerobically incubated in gas-tight bottles with N2O added to the headspace to induce N2O reduction. Pre-treatment included the removal of NO to prevent N2O production. Gas samples were collected regularly to determine the dynamics of N2O reduction, the time course of the isotopic signatures of residual N2O, and the associated isotope enrichment factors. To vary reduction rates and associated fractionation factors, several treatments were established including two levels of initial N2O concentration and anaerobic pre-incubation with or without addition of N2O. N2O reduction rates were affected by the soil type and initial N2O concentration. The ε18O and εbulk ranged between −13 and −20‰, and between −5 and −9‰, respectively. Both quantities were more negative in the Gleyic Podzol. The ε of the central N position (εα) was always larger than that of the peripheral N-position (εβ), giving εSP of −4 to −8‰. The ranges and variation patterns of ε were comparable with those from previous static incubation studies with soils. Moreover, we found a relatively constant ratio between ε18O and εbulk which is close to the default ratio of 2.5 that had been previously suggested. The fact that different soils exhibited comparable ε under certain conditions suggests that these values could serve to identify N2O reduction from the isotopic fingerprints of N2O emitted from any soil. Copyright © 2009 John Wiley & Sons, Ltd.