Combined oxygen (O) and nitrogen (N) stable isotope analyses are commonly used in the source determination of nitrate . The source and fate of are studied based on distinct O and N isotopic signatures (d18O and d15N) of various sources and isotopic effects during transformation processes, which differ between sources like fertilizer, atmospheric deposition, and microbial production (nitrification). Isotopic fractionation during production and consumption of further affects the d18O and d15N signal. Regarding the d18O in particular, biochemical O exchange between O from and H2O is implicitly assumed not to affect the d18O signature of . This study aimed to test this assumption in soil-based systems. In a short (24 h) incubation experiment, soils were treated with artificially 18O and 15N enriched . Production of from nitrification during the incubation would affect both the 18O and the 15N enrichment. Oxygen exchange could therefore be studied by examining the change in 18O relative to the 15N. In two out of the three soils, we found that the imposed 18O enrichment of the declined relatively more than the imposed enrichment. This implies that O exchange indeed affected the O isotopic signature of , which has important implications for source determination studies. We suggest that O exchange between and H2O should be taken into consideration when interpreting the O isotopic signature to study the origin and fate of in ecosystems. Highlights ¿ The oxygen isotopic signature of nitrate is commonly used to study its source and fate in ecosystems. ¿ The effect of oxygen exchange is in such studies generally not taken into account. ¿ We used a novel ‘enrichment ratio’ approach to evaluate changes the isotopic signature of NO3. ¿ Our study shows that oxygen exchange can affect the oxygen isotopic signature of nitrate in soils.