V0V1-ATPase is a proton-translocating ATPase responsible for acidification of eukaryotic intracellular compartments and for ATP synthesis in archaea and some eubacteria. We demonstrated recently the rotation of the central stalk subunits in V1, a catalytic sector of V0V1-ATPase (Imamura, H., Nakano, M., Noji, H., Muneyuki, E., Ohkuma, S., Yoshida, M., and Yokoyama, K. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 2312-2315), but the rotation of the proteolipid ring, a predicted counterpart rotor in the membrane V0 sector, has remained to be proven. V0V1-ATPase that retained sensitivity to N',N'-dicyclohexylcarbodiimide was isolated from Thermus thermophilus, immobilized onto a glass surface through the N termini of the A subunits of V1, and decorated with a bead attached to a proteolipid subunit of V0. Rotation of beads was observed in the presence of ATP, and direction of rotation was always counterclockwise viewed from the membrane side. The rotation proceeded at approximately 3.0 rev/s in average at 4 mm ATP and was abolished by N',N'-dicyclohexylcarbodiimide treatment. Thus, the rotation of the central stalk in V1 accompanies rotation of a proteolipid ring of V0 in the functioning V0V1-ATPase.