Abstract: The Saccharomyces cerevisiae vacuolar proton-translocating ATPase (V-ATPase) is composed of fourteen subunits distributed between a peripheral V(1) subcomplex and an integral membrane V(0) subcomplex. Genome-wide screens have led to the identification of the newest yeast V-ATPase subunit, Vma9p. Vma9p (subunit e) is a small hydrophobic protein that is conserved from fungi to animals. We demonstrate that disruption of yeast VMA9 results in the failure of V(1) and V(0) V-ATPase subunits to assemble onto the vacuole, and decreased levels of the subunit a isoforms Vph1p and Stv1p. We also show that Vma9p is an integral membrane protein, synthesized and inserted into the endoplasmic reticulum (ER), which then localizes to the limiting membrane of the vacuole. All V(0) subunits and V-ATPase assembly factors are required for Vma9p to efficiently exit the ER. In the ER, Vma9p and the V(0) subunits interact with the V-ATPase assembly factor Vma21p. Interestingly, the association of Vma9p with the V(0)-Vma21p assembly complex is disrupted with the loss of any single V(0) subunit. Similarly, Vma9p is required for V(0) subunits Vph1p and Vma6p to associate with the V{sub0)-Vma21p complex. In contrast, the proteolipids associate with Vma21p even in the absence of Vma9p. These results demonstrate that Vma9p is an integral membrane subunit of the yeast V-ATPase V(0) subcomplex, and suggest a model for the arrangement of polypeptides within the V(0) subcomplex.