Global analyses of protein phosphorylation require specific enrichment methods because of the typically low abundance of phosphoproteins. To date, immobilized metal-ion affinity chromatography (IMAC) for phosphopeptides has shown great promise for large-scale studies, but has a reputation for poor specificity. We investigated the potential of IMAC in combination with capillary liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) for the identification of plasma membrane phosphoproteins of Arabidopsis. Without chemical modification of peptides, over 75% pure phosphopeptides were isolated from plasma membrane digests and detected and sequenced by mass spectrometry. We present a scheme for two-dimensional peptide separation using strong anion exchange (SAX) chromatography prior to IMAC that both decreases the complexity of IMAC-purified phosphopeptides and yields a far greater coverage of monophosphorylated peptides. Among the identified sequences, six originated from different isoforms of the plasma membrane H+-ATPase and defined two previously unknown phosphorylation sites at the regulatory C-terminus. The potential for large-scale identification of phosphorylation sites on plasma membrane proteins will have wide-ranging implications for research in signal transduction, cell-cell communication and membrane transport processe