Abstract: There has been no previous indication that vacuolar ATPases (V-ATPases) require sphingolipids for function. Here we show, by using Saccharomyces cerevisiae sur4Delta and fen1Delta cells, that sphingolipids with a C26 acyl group are required for generating V1 domains with ATPase activity. Sphingolipids in sur4Delta cells contain C22 and C24 acyl groups instead of C26 acyl groups whereas about 30% of the sphingolipids in fen1Delta cells have C26 acyl groups and the rest have C22 and C24 acyl groups. sur4Delta cells have several phenotypes (vacuolar membrane ATPase, Vma-) that indicate a defect in the V-ATPase, and vacuoles purified from sur4Delta cells have little to no ATPase activity. These phenotypes are less pronounced in fen1Delta cells, consistent with the idea that the C26 acyl group in sphingolipids is necessary for V-ATPase activity. Other results show that the two V-ATPase domains, V1 and V0, are assembled and delivered to the vacuolar membrane in sur4Delta cells similar to wild-type cells. In vitro assembly studies show that V1 from sur4Delta cells associates with wild-type V0 but the complex lacks V-ATPase activity, indicating that V1 is defective. Reciprocal experiments with V0 from sur4Delta cells show that it is normal. We conclude that sphingolipids with a C26 acyl group are required for generating fully functional V1 domains.