The vacuolar ATPase (V-ATPase) is a multisubunit enzyme that acidifies intracellular organelles in eukaryotes. Similar to the F-type ATP synthase (FATPase), the V-ATPase is composed of two subcomplexes, V(1) and V(0). Hydrolysis of ATP in the V(1) subcomplex is tightly coupled to proton translocation accomplished by the V(0) subcomplex, which is composed of five unique subunits (a, d, c, c', and c"). Three of the subunits, subunit c (Vma3p), c' (Vma11p), and c" (Vma16p), are small highly hydrophobic integral membrane proteins called "proteolipids" that share sequence similarity to the F-ATPase subunit c. Whereas subunit c from the F-ATPase spans the membrane bilayer twice, the V-ATPase proteolipids have been modeled to have at least four transmembrane-spanning helices. Limited proteolysis experiments with epitope-tagged copies of the proteolipids have revealed that the N and the C termini of c (Vma3p) and c' (Vma11p) were in the lumen of the vacuole. Limited proteolysis of epitope-tagged c" (Vma16p) indicated that the N terminus is located on the cytoplasmic face of the vacuole, whereas the C terminus is located within the vacuole. Furthermore, a chimeric fusion between Vma16p and Vma3p, Vma16-Vma3p, was found to assemble into a fully functional V-ATPase complex, further supporting the conclusion that the C terminus of Vma16p resides within the lumen of the vacuole. These results indicate that subunits c and c' have four transmembrane segments with their N and C termini in the lumen and that c" has five transmembrane segments, with the N terminus exposed to the cytosol and the C terminus lumenal.