The plasma membrane H(+)-ATPase (Pmal) from Saccharomyces cerevisiae contains 14 histidine residues. The seven most highly conserved of these were selected as targets for oligonucleotide-directed, site-specific mutagenesis. Substitutions at H240, H488, H614, H686, and H914 with a variety of amino acid residues had no effect either on cell viability or on temperature and pH growth sensitivity. In contrast, substitutions at H701, located in the putative fifth membrane-spanning region, with Asp, Gln, or Arg were dominant lethal, indicating that H701 is essential for H(+)-ATPase activity. The mutations H285Q and H285R, but not H285E, located in the hydrophilic beta-stranded domain, were tolerated in normal growth conditions. Growth of H285Q mutants was sensitive to acid pH, indicating impaired in vivo ATPase activity. The H285Q and H285R mutants showed increased in vitro ATPase-specific activity, increased vanadate resistance, increased proton competition of vanadate sensitivity, accelerated ATP hydrolysis rates at a substrate concentration much lower than the Km, and slightly uncoupled proton pumping. The most reasonable hypothesis which would take into account these observations is that H285 would not be involved in the H+ transport process but rather in the E2 to E1 transition step of the ATP hydrolysis catalytic cycle.