The proteolipid domain of vacuolar H(+)-ATPase (V-ATPase) plays a major role in H+ transport in microvesicles and other acidic organelles. We have cloned the second human proteolipid of the V-ATPase (designated hATP6F), a homologue of the Saccharomyces cerevisiae proteolipid VMA16, which is an essential subunit of yeast V-ATPase. hATP6F is a hydrophobic protein with five putative transmembrane segments, having 61% amino acid identity and 83% similarity to the yeast protein, except in the N-terminus, and contains a conserved glutamic acid residue (Glu98) that is essential for H(+)-transporting activity. The gene for hATP6F (gene symbol, ATP6F), which consists of eight exons and spans approximately 3.5 kb, was isolated and mapped to human chromosome band 1p32.3 and the region 10.81 cR centromeric of the STS marker SHGC36789 (LOD = 6.75) by fluorescence in situ hybridization and radiation hybrid mapping, respectively. This is the first evidence in human of the existence of a second gene encoding a distinct V-ATPase proteolipid.