In most eukaryotic cells, vacuolar H(+)-ATPases (V-ATPases) are present primarily or exclusively in intracellular membrane compartments, functioning in the acidification of the endocytic and secretory vacuolar apparatus necessary for constitutive cell function. V-ATPases also participate in renal hydrogen ion secretion in both the proximal and distal nephron, residing at high concentrations on the plasma membrane, where they are regulated physiologically to maintain the acid-base balance of the organism. Recent experiments have begun to reveal how the kidney controls transcellular proton transport while still maintaining acidification of intracellular compartments. Control may occur by recruitment of proton pumps to or away from the plasma membrane. The proton-transporting plasma membrane of intercalated cells is a specialized apparatus that translocates the enzyme between an intracellular membrane pool and the plasma membrane in response to physiological stimuli. Regulation may also occur by changes in the kinetics of the V-ATPase. V-ATPases are a family of structurally similar enzymes which differ in the composition of specific subunits. Cytosolic regulatory enzymes present in renal cells may preferentially affect V-ATPases in selective membrane compartments.