Formation of a transmembrane electric potential coupled to ATP hydrolysis is demonstrated in chloroplast ATPase complex containing proteoliposomes. The ATP-induced signals were detected through absorbance changes of the membrane potential-responding dye oxonol VI. They were inhibited by the specific energy-transfer inhibitor, tentoxin and the ionophore valinomycin while stimulated by nigericin. Calibration of the transmembrane potential signal was possible by the application of a proton diffusion potential. The ATP-induced transmembrane potential was estimated to be 40–50 mV.