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For the advanced study of mitochondrial function, high-resolution respirometry is extended by fluorometric measurement of ATP production using the fluorophore Magnesium Green™ (MgG). A common problem with several fluorescent dyes is the inhibition of mitochondrial respiration. In the present study, a coupling control protocol was applied in combination with MgG to measure ATP production simultaneously with respiration for calculation of P»/O2 ratios. MgG at 1.1 µM did not affect respiration through the NADH-linked and succinate-linked pathways. Respiration was not inhibited in any of the coupling control states, hence coupling control efficiencies were not affected by MgG.