V1, a water-soluble portion of vacuole-type ATPase (V-ATPase), is an ATP-driven rotary motor, similar to F1-ATPase. Hydrolysis of ATP is coupled to unidirectional rotation of the central rotor D and F subunits relative to the A3B3 cylinder. In this study, we analyzed the rotation kinetics of V1 in detail. At low ATP concentrations, the D subunit rotated stepwise, pausing every 120°. The dwell time between steps revealed that V1 consumes one ATP per 120°step. V1 generated torque of Ϸ35 pN nm, slightly lower than the Ϸ46 pN nm measured for F1. Noticeably, the angles for both ATP cleavage and binding were apparently the same in V1, in sharp contrast to F1, which cleaves ATP at 80°posterior to the binding of ATP. Thus, the mechanochemical cycle of V1 has marked differences to that of F1.ATP synthase ͉ molecular motor ͉ single molecular analysis ͉ vacuole-type ATPase T he F 0 F 1 -ATP synthase (F)-and vacuole-type (V-type) ATPase͞ATP synthase-superfamily members utilize a rotary mechanism to perform their specific function (1-3). The cytoplasmic portion of F-and V-type ATPases (called F 1 and V 1 , respectively), responsible for ATP hydrolysis͞synthesis, is connected via the central rotor stalk and the peripheral stator stalk to the transmembrane portion (F 0 and V 0 ), housing the iontransporting pathway. The rotation of the central rotor subunits is a key feature of these enzymes in coupling ATP hydrolysis͞ synthesis with ion transport across the membrane. Isolated F 1 is, itself, a well characterized ATP-driven rotary motor that converts a chemical reaction (ATP hydrolysis) into mechanical rotation of the central-shaft subunits (1). Bacterial F 1 is composed of five different subunits with a composition of ␣ 3  3 ␥␦. The catalytic site of F 1 is formed at the interface between the ␣ and  subunits, with the majority of the catalytic residues residing in the  subunit. The minimum rotary unit of F 1 is the ␣ 3  3 ␥ subcomplex, in which the central ␥ subunit rotates within the ␣ 3  3 core by repeating a sequence of (at least) four processes: (i) binding of ATP to a catalytic  subunit, (ii) 80°rotation of the ␥ subunit, (iii) cleavage of ATP and͞or release of hydrolysis product(s), and (iv) 40°rotation of ␥ (4-7). It is notable that F 1 is a reversible motor. When the ␥ subunit of F 1 is rotated in the reverse direction by external force, ATP is synthesized from ADP and phosphate (8,9). It is believed that proton flow through F 0 drives the rotation of the proteolipid ring together with the ␥ subunit in the reverse direction to synthesize ATP in the cell. Recently, proton-powered reverse rotation of the ␥ subunit was observed for F 0 F 1 incorporated into liposomes (10).V-ATPases exist in the endomembranes of all eukaryotic cells and in the plasma membrane of some specific eukaryotic cells and are responsible for a variety of cellular functions (11). The enzyme has also been found in the plasma membrane of some bacteria (12)(13)(14). It is thought that V-ATPase is evolutionarily related to F-ATPase, because sever...