F_oF₁-ATP synthase (F_oF₁) synthesizes ATP in the F₁ portion when protons flow through F_o to rotate the shaft common to F₁ and F_o. Rotary synthesis in isolated F₁ alone has been shown by applying external torque to F₁ of thermophilic origin. Proton-driven ATP synthesis by thermophilic Bacillus PS3 F_oF₁ (TF_oF₁), however, has so far been poor in vitro, of the order of 1 s^-1 or less, hampering reliable characterization. Here, by using a mutant TF_oF₁ lacking an inhibitory segment of the ε-subunit, we have developed highly reproducible, simple procedures for the preparation of active proteoliposomes and for kinetic analysis of ATP synthesis, which was driven by acid–base transition and K⁺-diffusion potential. The synthesis activity reached ∼ 16 s^-1 at 30 °C with a Q10 temperature coefficient of 3–4 between 10 and 30 °C, suggesting a high level of activity at the physiological temperature of ∼ 60 °C. The Michaelis–Menten constants for the substrates ADP and inorganic phosphate were 13 μM and 0.55 mM, respectively, which are an order of magnitude lower than previous estimates and are suited to efficient ATP synthesis.