Summary A significant resistance to CO2 diffusion is imposed by mesophyll tissue inside leaves. Mesophyll resistance, rm (or its reciprocal, mesophyll conductance, gm), reduces the rate at which Rubisco can fix CO2, increasing the water and nitrogen costs of carbon acquisition. gm varies in proportion to the surface area of chloroplasts exposed to intercellular airspace per unit leaf area. It also depends on the thickness and effective porosity of the cell wall and the CO2 permeabilities of membranes. As no measurements exist for the effective porosity of mesophyll cell walls, and CO2 permeability values are too low to account for observed rates of CO2 assimilation, conclusions from modelling must be treated with caution. There is great variation in the mesophyll resistance per unit chloroplast area for a given cell wall thickness, which may reflect differences in effective porosity. While apparent gm can vary with CO2 and irradiance, the underlying conductance at the cellular level may remain unchanged. Dynamic changes in apparent gm arise for spatial reasons and because chloroplasts differ in their photosynthetic composition and operate in different light environments. Measurements of the temperature sensitivity of membrane CO2 permeability are urgently needed to explain variation in temperature responses of gm.