The CO2 concentration at the site of carboxylation inside the chloroplast stroma depends not only on the stomatal conductance, but also on the conductance of CO2 between substomatal cavities and the site of CO2 fixation. This conductance, commonly termed mesophyll conductance (gm), significantly constrains the rate of photosynthesis. Here we show that estimates of gm are influenced by the amount of respiratory and photorespiratory CO2 from the mitochondria diffusing towards the chloroplasts. This results in an apparent CO2 and oxygen sensitivity of gm that does not imply a change in intrinsic diffusion properties of the mesophyll, but depends on the ratio of mitochondrial CO2 release to chloroplast CO2 uptake. We show that this effect (1) can bias the estimation of the CO2 photocompensation point and non-photorespiratory respiration in the light; (2) can affect the estimates of ribulose 1·5-bisphosphate carboxylase/oxygenase (Rubisco) kinetic constants in vivo; and (3) results in an apparent obligatory correlation between stomatal conductance and gm. We further show that the amount of photo(respiratory) CO2 that is refixed by Rubisco can be directly estimated through measurements of gm.