The proteolipids of the vacuolar-type H+-ATPase (V-ATPase) are major components of the integral membrane sector. The vha-1 and vha-2 (vacuolar-type H+-ATPase) genes in Caenorhabditis elegans encode putative 16-kDa proteolipids and are tandemly localized on chromosome III. The vha-2 gene has three exons, whereas vha-1 has no introns. The deduced amino acid sequences of the two genes exhibit about 60% identity with the homologues from yeast, mouse, and cow. The mRNAs of both vha genes are trans-spliced to spliced leaders, suggesting that these genes constitute a polycistronic transcriptional unit. The vha-4 gene consists of four exons and is very similar to the yeast VMA16 gene that codes for the 23-kDa proteolipid. This is the first example of three distinct V-ATPase proteolipids being identified in higher eukaryotes. Northern blot and transgenic analyses show that the three vha genes may be highly expressed in the H-shaped excretory cell, rectum, and a pair of cells posterior to the anus. These results suggest that the V-ATPase activity may be important for exporting toxic compounds or metabolic wastes in this organism.