We compared the diffusion conductance to CO₂ from the intercellular air space to the chloroplasts (internal conductance (g _i)) between tobacco leaves acclimated to long-term drought (drought-acclimated (DA)) and those grown under sufficient irrigation (well-watered (WW)), and analysed the changes in g _i in relation to the leaf anatomical characteristics and a possible CO₂ transporter, aquaporin. The g _i, which was estimated by combined analyses of CO₂ gas exchange with chlorophyll fluorescence, in the DA plants was approximately half of that in the WW plants. The mesophyll and chloroplast surface areas exposing the intercellular air space, which potentially affect g _i, were not significantly different between the WW and DA plants. The amounts of plasma membrane aquaporins (PIP), immunochemically determined using radish PIP antibodies, were unrelated to g _i. After treatment with HgCl₂, an aquaporin inhibitor, the water permeability of the leaf tissues (measured as the weight loss of fully-turgid leaf disks without the abaxial epidermis in 1 m sorbitol) in WW plants decreased with an increase in HgCl₂ concentration. The g _i in the WW plants decreased to similar levels to the DA plants when the detached leaflets were fed with 0.5 mm HgCl₂. In contrast, both water permeability and g _i were insensitive to HgCl₂ treatments in DA plants. These results suggest that deactivation of aquaporins is responsible for the significant reduction in g _i observed in plants growing under long-term drought.