Barley (Hordeum vulgare L.) plants were grown hydroponically with or without inorganic phosphate (Pi) in the medium. Leaves were analyzed for the intercellular and the intracellular distribution of Pi. Most of the leaf Pi was contained in mesophyll cells; Pi concentrations were low in the xylem sap, the apoplast and in the cells of the epidermis. The vacuolar concentration of Pi in mesophyll cells depended on Pi availability in the nutrient medium. After infiltrating the intercellular space of leaves with solutions containing Pi, Pi was taken up by the mesophyll at rates higher than 2.5 μmol· (g fresh weight)(-1) · h(-1). Isolated mesophyll protoplasts did not possess a comparable capacity to take up Pi from the medium. Phosphate uptake by mesophyll protoplasts showed a biphasic dependence on Pi concentration. Uptake of Pi by Pi-deficient cells was faster than uptake by cells which had Pi stored in their vacuoles, although cytoplasmic Pi concentrations were comparable. Phosphate transport into isolated mesophyll vacuoles was dependent on their Pi content; it was stimulated by ATP. In contrast to the vacuolar Pi concentration, and despite different kinetic characteristics of the uptake systems for pi of the plasmalemma and the tonoplast, the cytoplasmic pi concentration was regulated in mesophyll cells within narrow limits under very different conditions of Pi availability in the nutrient medium, whereas vacuolar Pi concentrations varied within wide limits.