The yeast Saccharomyces cerevisiae vacuolar H(+)-ATPase (V-ATPase) is a multisubunit complex responsible for acidifying intracellular organelles and is highly regulated. One of the regulatory subunits, subunit H, is encoded by the VMA13 gene in yeast and is composed of two domains, the N-terminal domain (amino acids (aa) 1-352) and the C-terminal domain (aa 353-478). The N-terminal domain is required for the activation of the complex, whereas the C-terminal domain is required for coupling ATP hydrolysis to proton translocation (Liu, M., Tarsio, M., Charsky, C. M., and Kane, P. M. (2005) J. Biol. Chem. 280, 36978-36985). Experiments with epitope-tagged copies of Vma13p revealed that there is only one copy of Vma13p/subunit H per V-ATPase complex. Analysis of the N-terminal domain shows that the first 179 amino acids are not required for the activation and full function of the V-ATPase complex and that the minimal region of Vma13p/subunit H capable of activating the V-ATPase is aa 180-353 of the N-terminal domain. Subunit H is expressed as two splice variants in mammals, and deletion of 18 amino acids in yeast Vma13p corresponding to the mammalian subunit H beta isoform results in reduced V-ATPase activity and significantly lower coupling of ATPase hydrolysis to proton translocation. Intriguingly, the yeast Vma13p mimicking the mammalian subunit H beta isoform is functionally equivalent to Vma13p lacking the entire C-terminal domain. These results suggest that the mammalian V-ATPase complexes with subunit H splice variant SFD-alpha or SFD-beta are likely to have different activities and may perform distinct cellular functions.