Subunit 8 (Y8) is a component of the proton channel of yeast (Saccharomyces cerevisiae) mitochondrial ATP synthase (mtATPase), whose function in the complex remains to be precisely defined. Y8 variants truncated at residue 46 (Lys47-->STP), or in which each of three conserved C-terminal amino acid residues (Arg37, Arg42 and Lys47) were substituted with isoleucine, have defects in assembly and function. The additional positive charge substitution (Gln29-->Lys) was introduced into each of the variants to determine whether functional compensation for these defects could be achieved. In the case of the (Lys47-->STP) variant, the additional positive charge restored the ability of cells to grow on non-fermentable substrate. By contrast, for the (Lys47-->Ile) variant the additional positive charge did not confer any improvement in cellular growth rate compared to that of cells expressing the Lys47-->Ile substitution alone. For the (Arg42-->Ile) and (Arg37-->Ile) variants, the presence of the Gln29-->Lys substitution failed to restore growth of host cells lacking endogenous subunit 8 on non-fermentable substrate. However, use of an in vitro assembly assay revealed that, unlike their respective parents (Arg42-->Ile or Arg37-->Ile), the (Gln29-->Lys Arg42-->Ile) and (Gln29-->Lys Arg37-->Ile) variants assemble into mtATPase. Thus we conclude that Arg42 and Arg37 have a role in mtATPase function, in addition to being required for assembly of Y8 into mtATPase.