Leaf respiratory CO₂ is ¹³ C-enriched relative to leaf organic components in five species of C₃ plants

•  Here, we compared the carbon isotope ratios of leaf respiratory CO₂ (δ¹³ C_R ) and leaf organic components (soluble sugar, water soluble fraction, starch, protein and bulk organic matter) in five C₃ plants grown in a glasshouse and inside Biosphere 2. One species, Populus deltoides, was grown under three different CO₂ concentrations. •  The Keeling plot approach was applied to the leaf scale to measure leaf δ¹³ C_R and these results were compared with the δ¹³ C of leaf organic components. •  In all cases, leaf respiratory CO₂ was more ¹³ C-enriched than leaf organic components. The amount of ¹³ C enrichment displayed a significant species-specific pattern, but the effect of CO₂ treatment was not significant on P. deltoides. •  In C₃ plant leaves, ¹³ C-enriched respiratory CO₂ appears widespread. Among currently hypothesized mechanisms contributing to this phenomenon, non-statistical carbon isotope distribution within the sugar substrates seems most likely. However, caution should be taken when attempting to predict the δ¹³ C of leaf respiratory CO₂ at the ecosystem scale by upscaling the relationship between leaf δ¹³ C_R and δ¹³ C of leaf organic components.