Barley (Hordeum vulgare) protoplasts were incubated in darkness and in the light at saturating and limiting CO 2 concentrations. The protoplasts were fractioned by a membrane filtration technique which allows quenching of the metabolism by acidification within about 0.1 s and the ATP/ADP ratios in the cytasol, chloroplasts and mitochondria were determined. It is concluded that the cytosolic ATP/ADP ratio is considerably higher during photosynthesis at limiting CO 2 (which is the normal situation for a C 3 plant in air) compared to photosynthesis at saturating CO 2 or darkness.