The yeast vacuolar H+-ATPase (V-ATPase) is a multisubunit complex responsible for organelle acidification. The enzyme is structurally organized into two major domains: a peripheral domain (V1), containing the ATP binding sites, and an integral membrane domain (V0), forming the proton pore. Dissociation of the V1 and V0 domains inhibits ATP-driven proton pumping, and extracellular glucose concentrations regulate V-ATPase activity in vivo by regulating the extent of association between the V1 and V0 domains. To examine the mechanism of this response, we quantitated the extent of V-ATPase assembly in a variety of mutants with known effects on other glucose-responsive processes. Glucose effects on V-ATPase assembly did not involve the Ras-cyclic AMP pathway, Snf1p, protein kinase C, or the general stress response protein Rts1p. Accumulation of glucose 6-phosphate was insufficient to maintain or induce assembly of the V-ATPase, suggesting that further glucose metabolism is required. A transient decrease in ATP concentration with glucose deprivation occurs quickly enough to help trigger disassembly of the V-ATPase, but increases in cellular ATP concentrations with glucose readdition cannot account for reassembly. Disassembly was inhibited in two mutant enzymes lacking ATPase and proton pumping activities or in the presence of the specific V-ATPase inhibitor, concanamycin A. We propose that glucose effects on V-ATPase assembly occur by a novel mechanism that requires glucose metabolism beyond formation of glucose 6-phosphate and generates a signal that can be sensed efficiently only by a catalytically competent V-ATPase.