Abstract: The vacuolar H+-ATPase (V-ATPase) along with ion channels and transporters maintains vacuolar pH. V-ATPase ATP hydrolysis is coupled with proton transport and establishes an electrochemical gradient between the cytosol and vacuolar lumen for coupled transport of metabolites. Btn1p, the yeast homolog to human CLN3 that is defective in Batten disease, localizes to the vacuole. We previously reported that Btn1p is required for vacuolar pH maintenance and ATP-dependent vacuolar arginine transport. We report that extracellular pH alters both V-ATPase activity and proton transport into the vacuole of wild type Saccharomyces cerevisiae. V-ATPase activity is modulated through the assembly and disassembly of the V0 and V1 V-ATPase subunits located in the vacuolar membrane and on the cytosolic side of the vacuolar membrane, respectively. V-ATPase assembly is increased in yeast cells grown in high extracellular pH. In addition, at elevated extracellular pH, S. cerevisiae lacking BTN1 (btn1-), have decreased V-ATPase activity while proton transport into the vacuole remains similar to that for wild type. Thus, coupling of V-ATPase activity and proton transport in btn1- is altered. We show that down regulation of V-ATPase activity compensates the vacuolar pH imbalance for btn1- at early growth phases. We therefore propose that, Btn1p is required for tight regulation of vacuolar pH in order to maintain the vacuolar luminal content and optimal activity of this organelle, and that disruption in Btn1p function leads to a modulation of V-ATPase activity in order to maintain cellular pH homeostasis and vacuolar lumenal content.