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Previous work from our laboratory established that K + flux drives ATP synthesis in proteoliposomes and reconstituted ATP synthase in planar lipid membranes (Juhaszova et al., 2018, https://www.biorxiv.org/content/10.1101/355776v2). This data predicted that, in mitochondria, K + transport carried by F 1F o would share with H + ATP synthesis at a 3:1 ratio. To test this prediction, we investigated ATP synthesis and K +:H + stoichiometry in isolated rat heart mitochondria in the presence vs. absence of K + at 37 oC, matched osmolality (260 mOsm), pH 7.2. We quantified the oligomycin-sensitive O 2 consumption rate (OCR) with respirometry, ADP/O ratio with high-resolution respirometry, the proton motive force (PMF), ΔΨ m, ΔpH with radioactive tracers, and K + influx with a fluorescent indicator. Compared to its absence, the presence of K + produced 3.5-fold higher ATP synthesis flux accompanied by significant K + influx at 2.7:1 K +:H + stoichiometry and 2.65-fold higher OCR. Under similar conditions, we measured the PMF and its components during states 4 and 3 respiration. We found that, in the presence of K +, PMF decreased significantly (from 247±9 to 214±7mV, p<0.05) during the state 4◊3 transition, whereas no significant changes were found in the absence of K + (from 243±8 to 240±9 mV). The differential effect of K + on the PMF during the state 4◊3 transition was mainly driven by ΔΨ m (from 190±5 to 163±3 mV, p<0.0001) at similar ΔpH (from 59±5 to 54±6 mV). In contrast, in the absence of K +, the significant decrease (from 199±1 to 167±3 mV, p<0.0001) exhibited by ΔΨ m was concomitant with a significant increase in ΔpH (from 43±8 to 73±7 mV, p<0.05) resulting in 0.3 pH units increase in the matrix. Together, the data demonstrates that K + transport through F 1F o ATP synthase is driving the substantial observed increase in mitochondrial ATP synthesis at predicted stoichiometries.