In the vacuolar-type H+-ATPase (V-ATPase), highly hydrophobic subunits known as the proteolipids are components of the integral membrane V0 sector. Previously, we described the identification of three different proteolipid genes in Caenorhabditis elegans (Oka, T., Yamamoto, R., and Futai, M. (1997) J. Biol. Chem. 272, 24387-24392): vha-1 and vha-2 encoded 16-kDa subunits, and vha-4, a 23-kDa isoform. We report here that a third 16-kDa gene, vha-3, has been identified on chromosome IV. This is the first example in which four proteolipid genes have been found in a single organism. vha-2 and vha-3 exhibited 85% nucleotide identity within the open reading frames which encoded the identical amino acid sequence. Northern blot analysis indicated that all four genes were expressed in a similar pattern during the worm life cycle; however, studies with transgenic worms indicated that the vha-3 gene was expressed differently from other proteolipid genes in a cell-specific manner. These results implied that the isoforms of the proteolipids may be related to functional differences of V-ATPases in various cell types. Another new gene, vha-11, contained seven exons and was found to be located immediately downstream of vha-3. The two genes constitute a single transcriptional unit. The VHA-11 protein had 384 amino acids and shared strong sequence similarities with the C subunit, a component of the peripheral V1 sector of the V-ATPase, from yeast, bovine, and human. Expression of the vha-11 cDNA complemented a null mutation of VMA5, the yeast C subunit gene, thus demonstrating that vha-11 was the functional C subunit of C. elegans.