F 0 F 1 ATP synthases are the smallest rotary motors in nature and work as ATP factories in bacteria, plants and animals. Here we report on the first observation of intersubunit rotation in fully coupled single F 0 F 1 molecules during ATP synthesis or hydrolysis. We investigate the Na + -translocating ATP synthase of Propionigenium modestum specifically labeled by a single fluorophore at one c subunit using polarization-resolved confocal microscopy. Rotation during ATP synthesis was observed with the immobilized enzyme reconstituted into proteoliposomes after applying a diffusion potential, but not with a Na + concentration gradient alone. During ATP hydrolysis, stepwise rotation of the labeled c subunit was found in the presence of 2 mM NaCl, but not without the addition of Na + ions. Moreover, upon the incubation with the F 0 -specific inhibitor dicyclohexylcarbodiimide the rotation was severely inhibited.