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Mitochondrial contribution to photosynthetic metabolism during the transition from low light (25–100 μmol quanta m−2 s−1, limiting photosynthesis) to high light (500 μmol quanta m−2 s−1, saturating photosynthesis) was investigated in protoplasts from barley (Hordeum vulgare) leaves. After the light shift, photosynthetic oxygen evolution rate increased rapidly during the first 30–40 s and then declined up to 60–70 s after which the rate increased to a new steady-state after 80–110 s. Rapid fractionation of protoplasts was used to follow changes in sub-cellular distribution of key metabolites during the light shift and the activation state of chloroplastic NADP-dependent malate dehydrogenase (EC 1.1.1.82) was measured. Although oligomycin (an inhibitor of the mitochondrial ATP synthase) affected the metabolite content of protoplasts following the light shift, the first oxygen burst was not affected. However, the transition to the new steady-state was delayed. Rotenone (an inhibitor of mitochondrial complex I) had similar, but less pronounced effect as oligomycin. From the analysis of metabolite content and sub-cellular distribution we suggest that the decrease in oxygen evolution following the first oxygen burst is due to phosphate limitation in the chloroplast stroma. For the recovery the control protoplasts can utilize ATP supplied by mitochondrial oxidative phosphorylation to quickly overcome the limitation in stromal phosphate and to increase the content of Calvin cycle metabolites. The oligomycin-treated protoplasts were deficient in cytosolic ATP and thereby unable to support Calvin cycle operation. This resulted in a delayed capacity to adjust to a sudden increase in light intensity.