The yeast vacuolar proton-translocating ATPase (V-ATPase) is the best characterized member of the V-ATPase family. Biochemical and genetic screens led to the identification of a large number of genes in yeast, designated VMA, encoding proteins required to assemble a functional V-ATPase. A total of thirteen genes encode subunits of the final enzyme complex. In addition to subunit-encoding genes, we have identified three genes that code for proteins that are not part of the final V-ATPase complex yet required for its assembly. We refer to these nonsubunit Vma proteins as assembly factors, since their function is dedicated to assembling the V-ATPase. The assembly factors, Vma12p, Vma21p, and Vma22p are localized to the endoplasmic reticulum (ER) and aid the assembly of newly synthesized V-ATPase subunits that are translocated into the ER membrane. At least two of these proteins, Vma12p and Vma22p, function together in an assembly complex and interact directly with nascent V-ATPase subunits.