Variation in the oxygen isotope composition of within-canopy CO₂ has potential to allow partitioning of the ecosystem respiratory flux into above- and below-ground components. Recent theoretical work has highlighted the sensitivity of the oxygen isotope composition of leaf-respired CO₂ (δ_Rl) to nocturnal stomatal conductance. When the one-way flux model was tested on Ricinus communis L. large enrichments in δ_Rl were observed. However, most species for which the isotope flux partitioning technique has been or would be applied (i.e. temperate tree species) are much more conservative users of water than R. communis. So, high stomatal conductance and very high enrichment of δ_Rl observed may not be typical for temperate tree species. Using existing gas-exchange measurements on six temperate tree species, we demonstrate significant water loss through stomata for all species (i.e. statistically significantly greater than cuticular loss alone) at some time for some leaves during the night. δ_Rl values predicted by the one-way flux model revealed that δ_Rl might be very much more enriched than when the net flux alone is considered, particularly close to sunrise and sunset. Incorporation of the one-way flux model into ecosystem respiration partitioning studies will affect model outputs and interpretation of variation in the oxygen isotope composition of atmospheric CO₂.