The yeast vacuolar membrane proton-translocating ATPase (V-ATPase) is a multisubunit complex comprised of peripheral catalytic, and integral membrane domains. At least eight proteins cofractionate with purified preparations of the enzyme including 100-, 69-, 60-, 42-, 36-, 32-, 27-, and 17-kDa polypeptides (Kane, P.M., Yamashiro, C.T., and Stevens, T.H. (1989a) J. Biol. Chem. 264, 19236-19244). We took a reverse genetic approach to clone the structural gene for the 36-kDa subunit of the V-ATPase, VMA6, vma6 null mutants displayed growth characteristics typical of other vma mutants including sensitivity to media buffered at neutral pH or media containing 100 mM Ca2+. Vacuolar acidification was defective in vma6 cells and isolated vacuolar membrane preparations contained no detectable V-ATPase activity. The VMA6 gene encodes a hydrophilic polypeptide of 345 amino acids (predicted molecular mass 39.8-kDa). We present evidence that the VMA6 gene product (Vma6p) is a non-integral membrane component of the membrane pore domain and is required for V-ATPase complex assembly. Vma6p was removed from wild type vacuolar membranes by strong chaotropic agents such as alkaline Na2CO3 or 5M urea, which did not remove integral membrane polypeptides. In yeast cells lacking the integral membrane portion of the V-ATPase complex, Vma6p was unable to stably associate with vacuolar membranes. Conversely, in mutants specifically lacking Vma6p, components of the V-ATPase integral membrane domain were destabilized, and peripheral subunits failed to assemble onto vacuolar membranes. These results are discussed in the context of a developing model for V-ATPase assembly in yeast.