The ¹⁸O enrichment (Δ¹⁸O) of cellulose (Δ¹⁸O_Cel) is recognized as a unique archive of past climate and plant function. However, there is still uncertainty regarding the proportion of oxygen in cellulose (p_ex) that exchanges post‐photosynthetically with medium water of cellulose synthesis. Particularly, recent research with C₃ grasses demonstrated that the Δ¹⁸O of leaf sucrose (Δ¹⁸O_Suc, the parent substrate for cellulose synthesis) can be much higher than predicted from daytime Δ¹⁸O of leaf water (Δ¹⁸O_LW), which could alter conclusions on photosynthetic versus post‐photosynthetic effects on Δ¹⁸O_Cel via p_ex. Here, we assessed p_ex in leaves of perennial ryegrass (Lolium perenne) grown at different atmospheric relative humidity (RH) and CO₂ levels, by determinations of Δ¹⁸O_Cel in leaves, Δ¹⁸O_LGDZW (the Δ¹⁸O of water in the leaf growth‐and‐differentiation zone) and both Δ¹⁸O_Suc and Δ¹⁸O_LW (adjusted for ε_bio, the biosynthetic fractionation between water and carbohydrates) as alternative proxies for the substrate for cellulose synthesis. Δ¹⁸O_LGDZW was always close to irrigation water, and p_ex was similar (0.53 ± 0.02 SE) across environments when determinations were based on Δ¹⁸O_Suc. Conversely, p_ex was erroneously and variably underestimated (range 0.02–0.44) when based on Δ¹⁸O_LW. The photosynthetic signal fraction in Δ¹⁸O_Cel is much more constant than hitherto assumed, encouraging leaf physiological reconstructions.

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