This chapter discusses the isolation and functional reconstitution of the vacuolar H+-ATPase. Vacuolar proton-translocating ATPases acidify the lumen of internal organelles of eukaryotic cells, including those of the endocytic pathway, the Golgi network, lysosomes, vacuoles, and secretory vesicles. Much of the information concerning plant V-type ATPases has come from studying the transport properties of the enzyme in native vesicles and the protein structure and ATP hydrolytic activity of the detergent solubilized and purified H+-ATPases. The incorporation of purified H+-ATPases into sealed phospholipid vesicles, referred to as “reconstitution,” has now enabled studies of the H+-transport activity of plant enzymes of this class in the absence of other ion-conducting membrane proteins. The reconstitution procedure presented here has been used to study the subunit composition and regulation of the oat root V-type H+-ATPases and may have further utility in the identification and characterization of additional H+-ATPase complexes from other plant tissues or membrane fractions.