FoF1-ATP synthase (FoF1) is a rotary molecular motor that reversibly catalyzes ATP hydrolysis/synthesis reaction coupled with the proton translocation across the cellmembrane. Rotationmechanism of FoF1 (or isolated F1 part) driven by ATP hydrolysis has been extensively studied by single molecule techniques. However, rotation driven by protonmotive force, generated by the membrane potential and the difference in proton concentration across the membrane, has not been directly observed yet. Although we have addressed this issue using the planar membrane method (Ide and Yanagida, 1999), even the ATP-driven rotation of FoF1 embedded in planar membrane has been rarely observed. In this study, we tried supported membrane method as an alternative. FoF1 from Escherichia coli was reconstituted into the large supported membrane (>10 mm in diameter) formed on the NiNTAmodified coverglass, and immobilized via histidine-tags introduced into c-ring of Fo. Rotation was observed by streptavidin-coated 200nm latex beads attached to the biotinylated b subunits of F1. The number of rotating particles (~5) found in a single observation chamber increased significantly as compared with that found in the planar membrane (<0.1). Furthermore, the rotational speed (>10Hz) was much faster than that observed in the planar membrane (<1Hz) at high ATP concentration. These results indicate that planar membrane, formed in the presence of an organic solvent such as squalene, may interfere with FoF1 rotation, presumably due to its thickness. To drive the reverse rotation of FoF1, we are trying to apply protonmotive force across the supported membrane.