The yeast plasma membrane H+-ATPase is activated in vivo by glucose metabolism, and previous deletion analysis has shown the C-terminus of the enzyme to be involved in this regulation. Site-directed mutagenesis demonstrates that Arg909 and Thr912 at the C-terminus are important for the increase in Vmax of the ATPase induced by glucose. Other changes in kinetic parameters induced by glucose are largely independent of these amino acids. Arg909 and Thr912 form a potential phosphorylation site for calmodulin-dependent multiprotein kinase. A double mutation of Ser911 and Thr912 to Ala results in no cell growth in glucose medium and greatly reduced activation of the ATPase by glucose. Growth and activity are restored by a third mutation (Ala547----Val) at the catalytic domain, providing genetic evidence for domain interaction.