The proton pump ATPase (H(+)-ATPase) of the plant plasma membrane is regulated by an autoinhibitory C-terminal domain, which can be displaced by phosphorylation of the penultimate Thr residue and the subsequent binding of 14-3-3 proteins. We performed a mass spectrometric analysis of PMA2 (plasma membrane H(+)-ATPase isoform 2) isolated from Nicotiana tabacum suspension cells and identified two new phosphorylated residues in the enzyme 14-3-3 protein binding site: Thr(931) and Ser(938). When PMA2 was expressed in Saccharomyces cerevisiae, mutagenesis of each of these two residues into Asp prevented growth of a yeast strain devoid of its own H(+)-ATPases. When the Asp mutations were individually introduced in a constitutively activated mutant of PMA2 (E14D), they still allowed yeast growth but at a reduced rate. Purification of His-tagged PMA2 showed that the T931D or S938D mutation prevented 14-3-3 protein binding, although the penultimate Thr(955) was still phosphorylated, indicating that Thr(955) phosphorylation is not sufficient for full enzyme activation. Expression of PMA2 in an N. tabacum cell line also showed an absence of 14-3-3 protein binding resulting from the T931D or S938D mutation. Together, the data show that activation of H(+)-ATPase by the binding of 14-3-3 proteins is negatively controlled by phosphorylation of two residues in the H(+)-ATPase 14-3-3 protein binding site. The data also show that phosphorylation of the penultimate Thr and 14-3-3 binding each contribute in part to H(+)-ATPase activation.