Regulation of the contents and volume of vacuoles in plant cells depends on the coordinated activities of transporters and channels located in the tonoplast (vacuolar membrane). The three major components of the tonoplast are two proton pumps, the vacuolar H+-ATPase (V-ATPase) and H+-pyrophosphatase (V-PPase), and aquaporins. The tertiary structure of the V-ATPase complex and properties of its subunits have been characterized by biochemical and genetic techniques. These studies and a comparison with the F-type ATPase have enabled estimation of the dynamics of V-ATPase activity during catalysis. V-PPase, a simple proton pump, has been identified and cloned from various plant species and other organisms, such as algae and phototrophic bacteria, and functional motifs of the enzyme have been determined. Aquaporin, serving as the water channel, is the most abundant protein in the tonoplast in most plants. A common molecular architecture of aquaporins in mammals and plants has been determined by two-dimensional crystallographic analysis. Furthermore, recent molecular biological studies have revealed several other types of tonoplast transporters, such as the Ca2+-ATPase, Ca2+/H+ antiporter and Na+/H+ antiporter. Many other transporters and channels in the tonoplast remain to be identified; their activities have already been detected. This review presents an overview of the field and discusses recent findings on the tonoplast protein components that have been identified and their physiological consequences.