The 18O enrichment (Δ 18O) of leaf water affects the Δ 18O of photosynthetic products such as sucrose, generating an isotopic archive of plant function and past climate. However, uncertainty remains regarding how leaf water compartmentation between photosynthetic and non-photosynthetic tissue affects the relationship between Δ 18O of bulk leaf water (Δ 18O LW) and leaf sucrose (Δ 18O Sucrose). We grew Lolium perenne (a C 3 grass) in mesocosm-scale, replicated experiments with daytime relative humidity (RH 50 or 75%) and CO 2 level (200, 400 or 800 μmol mol -1) as factors, and determined Δ 18O LW, Δ 18O Sucrose and morpho-physiological leaf parameters, including transpiration ( E leaf), stomatal conductance ( g s) and mesophyll conductance to CO 2 ( g m). The Δ 18O of photosynthetic medium water (Δ 18O SSW) was estimated from Δ 18O Sucrose and the equilibrium fractionation between water and carbonyl groups (ε bio). Δ 18O SSW was well predicted by theoretical estimates of leaf water at the evaporative site (Δ 18O e) with adjustments that correlated with gas exchange parameters ( g s or total conductance to CO 2). Isotopic mass balance and published work indicated that non-photosynthetic tissue water was a large fraction (~0.53) of bulk leaf water. Δ 18O LW was a poor proxy for Δ 18O Sucrose, mainly due to opposite Δ 18O responses of non-photosynthetic tissue water (Δ 18O non-SSW) relative to Δ 18O SSW, driven by atmospheric conditions.