The function of the amino- and carboxyl-terminal domains of the yeast plasma membrane H+-ATPase have been investigated by constructing deletions in vitro and selectively expressing the mutant enzymes in vivo. The first 27 amino acids are dispensable but deletion of a further 33 amino acids greatly decreases the appearance of the enzyme in the plasma membrane. Membrane localization is also prevented by carboxyl-terminal deletions which include the last hydrophobic stretch, but the last 46 amino acids of the ATPase are not required. Removal of the last 11 amino acids produces an enzyme in glucose-starved cells with the kinetic parameters of the wild-type ATPase activated by glucose fermentation. This region seems to constitute a regulatory domain.