Anaerobic incubations of upland and wetland temperate forest soils from the same watershed were conducted under different moisture and temperature conditions. Rates of nitrous oxide (N 2 O) production by denitrification of nitrate (NO3-) and the stable isotopic composition of the N 2 O (δ 15 N, δ 18 O) were measured. In all soils, N 2 O production increased with elevated temperature and soil moisture. At each temperature and moisture level, the rate of N 2 O production in the wetland soil was greater than in the upland soil. The 15 N isotope effect (ε) (product − substrate) ranged from −20‰ to −29‰. These results are consistent with other published estimates of 15 N fractionation from both single species culture experiments and soil incubation studies from different ecosystems.A series of incubations were conducted with 18 O-enriched water (H 2 O) to determine if significant oxygen exchange (O-exchange) occurred between H 2 O and N 2 O precursors during denitrification. The exchange of H 2 O–O with nitrite (NO2-) and/or nitric oxide (NO) oxygen has been documented in single organism culture studies but has not been demonstrated in soils prior to this study. The fraction of N 2 O–O derived from H 2 O–O was confined to a strikingly narrow range that differed between soil types. H 2 O–O incorporation into N 2 O produced from upland and wetland soils was 86% to 94% and 64% to 70%, respectively. Neither the temperature, soil moisture, nor the rate of N 2 O production influenced the magnitude of O-exchange. With the exception of one treatment, the net 18 O isotope effect (ε net ) (product–substrate) ranged from +37‰ to +43‰.Most previous studies that have reported 18 O isotope effects for denitrification of NO3- to N 2 O have failed to account for the effect of oxygen exchange with H 2 O. When high amounts of O-exchange occur after fractionation during reductive O-loss, the 18 O-enrichment is effectively lost or diminished and δ 18 O–N 2 O values will be largely dictated by δ 18 O–H 2 O values and subsequent fractionation. The process and extent of O-exchange, combined with the magnitude of oxygen isotope fractionation at each reduction step, appear to be the dominant controls on the observed oxygen isotope effect. In these experiments, significant oxygen isotope fractionation was observed to occur after the majority of water O-exchange. Due to the importance of O-exchange, the net oxygen isotope effect for N 2 O production in soils can only be determined using δ 18 O–H 2 O addition experiments with δ 18 O–H 2 O close to natural abundance.The results of this study support the continued use of δ 15 N–N 2 O analysis to fingerprint N 2 O produced from the denitrification of NO3-. The utilization of 18 O/ 16 O ratios of N 2 O to study N 2 O production pathways in soil environments is complicated by oxygen exchange with water, which is not usually quantified in field studies. The oxygen isotope fractionation observed in this study was confined to a narrow range, and there was a clear difference in water O-exchange between soil types regardless of temperature, soil moisture, and N 2 O production rate. This suggests that 18 O/ 16 O ratios of N 2 O may be useful in characterizing the actively denitrifying microbial community.