Pma1 H+-ATPase is the primary proton pump in the plasma membrane of the yeast Saccharomyces cerevisiae. It generates an electrochemical proton gradient, thus providing energy for secondary solute transport systems. The enzyme contains nine cysteines, three (Cys148, Cys312, and Cys867) in transmembrane segments and the rest (Cys221, Cys376, Cys409, Cys472, Cys532, and Cys569) in the cytosolic parts of the molecule. Although individually they are not essential for the functioning of the ATPase, substitution of all of them leads to the loss of enzyme activity and impairment of biogenesis. By means of site-directed mutagenesis combined with other molecular-biological and biochemical methods, this work defines different combinations of minimal cysteine content that are consistent with ATPase function.