The vacuolar proton-translocating ATPase is the principal energization mechanism that enables the yeast vacuole to perform most of its physiological functions. We have undertaken an examination of subunit-subunit interactions and assembly states of this enzyme. Yeast two-hybrid data indicate that Vma1p and Vma2p interact with each other and that Vma4p interacts with itself. Three-hybrid data indicate that the Vma4p self-interaction is stabilized by both Vma1p and Vma2p. Native gel electrophoresis reveals numerous partial complexes not previously described. In addition to a large stable cytoplasmic complex seen in wild-type, Deltavma3 and Deltavma5 strains, we see partial complexes in the Deltavma4 and Deltavma7 strains. All larger complexes are lost in the Deltavma1, Deltavma2, and Deltavma8 strains. We designate the large complex seen in wild-type cells containing at least subunits Vma1p, Vma2p, Vma4p, Vma7p, and Vma8p as the definitive V1 complex.