We have examined the possible role of leaf cytosolic hexoses and the expression of mannitol metabolism as mechanisms that may affect the repression of photosynthetic capacity when plants are grown at 1000 versus 380 [mu]L L-1 CO2. In plants grown at high CO2, leaf ribulose-1,5-bisphosphate carboxylase/oxygenase content declined by [greater than or equal to]20% in tobacco (Nicotiana sylvestris) but was not affected in the mannitol-producing species snapdragon (Antirrhinum majus) and parsley (Petroselinum hortense). In the three species mesophyll glucose and fructose at midday occurred almost entirely in the vacuole (>99%), irrespective of growth CO2 levels. The estimated cytosolic concentrations of glucose and fructose were [less than or equal to]100 [mu]M. In the three species grown at high CO2, total leaf carbohydrates increased 60 to 100%, but mannitol metabolism did not function as an overflow mechanism for the increased accumulation of carbohydrate. In both snapdragon and parsley grown at ambient or high CO2, mannitol occurred in the chloroplast and cytosol at estimated midday concentrations of 0.1 M or more each. The compartmentation of leaf hexoses and the metabolism of alternate carbohydrates are further considered in relation to photosynthetic acclimation to high levels of CO2.