The content of ribulose-l,5-bisphosphate carboxylase/oxygenase (Rubisco) (E-t; EC 4.1.1.39) measured in different-aged leaves of sunflower (Helianthus annuus) and other plants grown under different light intensities, varied from 2 to 75 mu mol active sites m(-2) Mesophyll conductance (mu) was measured under 1.5% O-2, as well as postillumination CO2 uptake (assimilatory charge, a gas-exchange measure of the ribulose-1,5-bisphosphate pool). The dependence of mu on E-t saturated at E-t = 30 mu mol active sites m(-2) and mu = 11 mm s(-1) in high-light-grown leaves. In low-light-grown leaves the dependence tended toward saturation at similar E-t but reached a mu of only 6 to 8 mm s(-1). mu was proportional to the assimilatory charge, with the proportionality constant (specific carboxylation efficiency) between 0.04 and 0.075 mu M-1 s(-1). Our data show that the saturation of the relationship between E-t and mu is caused by three limiting components: (a) the physical diffusion resistance (a minor limitation), (b) less than full activation of Rubisco (related to Rubisco activase and the slower diffusibility of Rubisco at high protein concentrations in the stroma), and (c) chloroplast metabolites, especially S-phosphoglyceric acid and free inorganic phosphate, which control the reaction kinetics of ribulose-1,5-bisphosphate carboxylation by competitive binding to active sites.