V-ATPases (vacuolar ATPases) are responsible for acidification of intracellular compartments and, in certain cases, proton transport across the plasma membrane of eukaryotic cells. They are composed of a catalytic V1 sector, in which ATP hydrolysis takes place, and the Vo sector, which functions in proton conduction. The best established mechanism for regulating the V-ATPase activity in vivo involves reversible dissociation of the V1 and Vo domains, in which subunit C is intimately involved. In the last year, impressive progress has been made in elucidating the structure of the C subunit and its arrangement inside the V-ATPase. Nucleotide occupancy by subunit C, followed by conformational changes of this subunit has shed light on the mechanism of V-ATPase regulation.