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Intact mesophyll protoplasts were isolated from barley (C3) and sorghum (C4) leaves for the measurements of osmotic volume change and metabolite uptake. We first investigated whether the silicone oil layer filtering centrifugation method could be applied to the protoplasts. The density of the silicone oil was optimized (p=1.026) and 0.5M betaine was chosen as an osmoticum in the protoplast suspending medium. By using ["C] sorbitol and ["C] inulin as the marker of the medium carried over into the pellet, protoplast osmotic or internal volume was estimated to be 200-300 pl (mg Chl)-l, with the medium space in the pellet of 8-15 pl (mg Chl)-l. Lowering of the osmotic pressure of the medium induced protoplast swelling as expected. Light also induced swelling. Using this system, we could detect light-enhanced uptake of ascorbate, glutamate and pyruvate in both barley and sorghum protoplasts. Pyruvate uptake was far higher in barley than in sorghum and inhibited by various inhibitors, showed saturation kinetics and, therefore, seemed to be mediated by a translocator protein.