ABSTRACT Vacuolar-type ATPases (V-ATPases) are proton-translocating enzymes that occur in the endomembranes of all eukaryotes and in the plasma membranes of many eukaryotes. They are multisubunit, heteromeric proteins composed of two structural domains, a peripheral, catalytic V1 domain and a membrane-spanning Vo domain. Both the multitude of locations and the heteromultimeric structure make it likely that the expression and the activity of V-ATPases are regulated in various ways. Regulation of gene expression encompasses control of transcription as well as control at the post-transcriptional level. Regulation of enzyme activity encompasses many diverse mechanisms such as disassembly/reassembly of V1 and Vo domains, oxidation of SH groups, control by activator and inhibitor proteins or by small signalling molecules, and sorting of the holoenzyme or its subunits to target membranes.