The crystal structure of subunit F of vacuole-type ATPase/synthase (prokaryotic V-ATPase) was determined to of 2.2 A resolution. The subunit reveals unexpected structural similarity to the response regulator proteins that include the Escherichia coli chemotaxis response regulator CheY. The structure was successfully placed into the low-resolution EM structure of the prokaryotic holo-V-ATPase at a location indicated by the results of crosslinking experiments. The crystal structure, together with the single-molecule analysis using fluorescence resonance energy transfer, showed that the subunit F exhibits two conformations, a 'retracted' form in the absence and an 'extended' form in the presence of ATP. Our results postulated that the subunit F is a regulatory subunit in the V-ATPase.