In recent years there has been growing interest in the posttranslational regulation of P-type ATPases by protein kinase-mediated phosphorylation. Pma1 H+-ATPase, which is responsible for H+-dependent nutrient uptake in yeast (Saccharomyces cerevisiae), is one such example, displaying a rapid 5- to 10-fold increase in activity when carbon-starved cellsare exposed to glucose. Activation has been linked to Ser/Thr phosphorylation in the carboxy terminal tail of the ATPase, but the specific phosphorylation sites have not previously been mapped. The present study has used nanoflow high-pressure liquid chromatography (nHPLC) coupled with electrospray electron transfer dissociation (ETD) tandem mass spectrometry to identify Ser-911 and Thr-912 as two major phosphorylation sites that are clearly related to glucose activation. In carbon-starved cells with low Pma1 activity, peptide 896–918, which was derived from the carboxy terminus upon Lys-C proteolysis, was found to be singly phosphorylated at Thr-912, while in glucose metabolizing cells with high ATPase activity, the same peptide was doubly phosphorylated at Ser-911 and Thr-912. Reciprocal 14N/15N metabolic labeling of cells was used to measure the relative phosphorylation levels at the two sites. The addition of glucose to carbon-starved cells led to a three-fold reduction in the singly phosphorylated form and an eleven-fold increase in the doubly phosphorylated form. These results point to a mechanism in which the stepwise phosphorylation of two tandemly positioned residues near the carboxy terminus mediates glucose-dependent activation of the H+-ATPase.