The effect of transpiration rate on internal assimilation of CO2 released from respiring cells has not previously been quantified. In this study, detached branches of Populus deltoides were allowed to take up (CO2)-C-13-labelled solution at either high (high label, HL) or low (low label, LL) (CO2)-C-13 concentrations. The uptake of the (CO2)-C-13 label served as a proxy for the internal transport of respired CO2, whilst the transpiration rate was manipulated at the leaf level by altering the vapour pressure deficit (VPD) of the air. Simultaneously, leaf gas exchange was measured, allowing comparison of internal CO2 assimilation with that assimilated from the atmosphere. Subsequent C-13 analysis of branch and leaf tissues revealed that woody tissues assimilated more label under high VPD, corresponding to higher transpiration, than under low VPD. More C-13 was assimilated in leaf tissue than in woody tissue under the HL treatment, whereas more C-13 was assimilated in woody tissue than in leaf tissue under the LL treatment. The ratio of (CO2)-C-13 assimilated from the internal source to CO2 assimilated from the atmosphere was highest for the branches under the HL and high VPD treatment, but was relatively small regardless of VPDlabel treatment combination (up to 1.9%). These results showed that assimilation of internal CO2 is highly dependent on the rate of transpiration and xylem sap [CO2]. Therefore, it can be expected that the relative contribution of internal CO2 recycling to tree carbon gain is strongly dependent on factors controlling transpiration, respiration, and photosynthesis.