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The classical view of oxidative phosphorylation is that a proton motive force (PMF) generated by the respiratory chain complexes fuels ATP synthesis via ATP synthase. Yet, under glycolytic conditions, ATP synthase in its reverse mode also can contribute to the PMF. Here, we dissected these two functions of ATP synthase and the role of its inhibitory factor 1 (IF1) under different metabolic conditions. pH profiles of mitochondrial sub‐compartments were recorded with high spatial resolution in live mammalian cells by positioning a pH sensor directly at ATP synthase's F1 and FO subunits, complex IV and in the matrix. Our results clearly show that ATP synthase activity substantially controls the PMF and that IF1 is essential under OXPHOS conditions to prevent reverse ATP synthase activity due to an almost negligible ΔpH. In addition, we show how this changes lateral, transmembrane, and radial pH gradients in glycolytic and respiratory cells. SYNOPSIS: Mitochondrial F1FO ATP synthase synthesizes or hydrolyses ATP depending on the metabolic conditions and controlled by IF1. Analysis of pH values using local pH sensors at ATP synthase and ATP synthase subcomplexes discloses these activities, which essentially contribute to the establishment of radial, transmembrane, and lateral pH gradients in cristae. Complementing the recent report on intramitochondrial inhomogeneity of ΔΨm these findings provide a new perspective on the heterogeneity of protonic energy coupling. The ΔpH along and across the inner mitochondrial membrane is not homogeneous.Local pH at cristae rims is controlled by F1FO ATP synthase.Under OXPHOS conditions, the pH difference between FO and F1 of active ATP synthase is almost negligible (1.2 proton versus 1 proton equivalent).Consequently, IF1 is required to prevent the onset of ATP hydrolysis also under OXPHOS conditions. [ABSTRACT FROM AUTHOR]