The contribution of mitochondrial oxidation of photorespiratory and respiratory substrates to subcellular energy and redox balance was investigated in leaf protoplasts of barley (Hordeum vulgare L.). The ATP/ADP ratios (indicating the energy balance) in chloroplasts and in tine extrachloroplast compartment were highest in the light in limiting CO2 (photorespiratory conditions), and they drastically increased after illumination if plants were pre-incubated in darkness for 24 hours. After illumination, the ATP/ADP ratio rapidly decreased in chloroplasts. The NADPH/NADP ratio (as an indicator of redox balance) in chloroplasts declined rapidly during the first seconds of darkness, then slowly increased. In limiting CO2, the ratio decreased more slowly during the first minute of darkness corresponding to post-illumination respiratory burst (PIE). During this period, the activation state of chloroplast NADP-malate dehydrogenase was higher in limiting CO2 than in saturating CO2. However, during the light-enhanced dark respiration (LEDR) period, following PIE, there were no differences in subcellular NADPH/NADP ratios in saturating and limiting CO2. A decline in malate and citrate concentrations in protoplasts and activation of mitochondrial NAD-malic enzyme were revealed during LEDR. The results presented highlight the importance of glycine oxidation in mitochondria in energization of the cytosol and chloroplasts and in maintaining redox balance in the light and during the first minute after illumination. And further, they show non-photorespiratory origin of LEDR.