Phosphatidylinositol 4,5-biphosphate (PIP₂) modulates the function of numerous ion transporters and channels, as well as cell signaling and cytoskeletal proteins. To study PIP₂ levels of cells without radiolabeling, we have developed a new method to quantify anionic phospholipid species. Phospholipids are extracted and deacylated to glycero-head groups, which are then separated by anion-exchange HPLC and detected by suppressed conductivity measurements. The major anionic head groups can be quantified in single runs with practical detection limits of about 100 pmol, and the D3 isoforms of phosphatidylinositol phosphate (PIP) and PIP₂ are detected as shoulder peaks. In HeLa, Hek 293 and COS cells, as well as intact heart, PIP₂ amounts to 0.5 to 1.5% of total anionic phospholipid (10 to 30 μmol/liter cell water or 0.15 to 0.45 nmol/mg protein). In cell cultures, overexpression of Type I PIP5-kinase specifically increases PIP₂, whereas overexpression of Type II PI4-kinase can increase both PIP and PIP₂. Phosphatidylinositol 3,4,5-trisphosphate (PIP₃) and the D3 isomers of PIP₂ are detected after treatment of cells with pervanadate; in yeast, overexpression of a phosphatidylinositol 3-kinase (VPS34) specifically increases phosphatidylinositol 3-phosphate (PI3P). Using isolated cardiac membranes, lipid kinase and lipid phosphatase activities can be monitored with the same methods. Upon addition of ATP, PIP increases while PIP₂ remains low; exogenous PIP₂ is rapidly degraded to PIP and phosphatidylinositol (PI). In summary, the HPLC methods described here can be used to probe multiple aspects of phosphatidylinositide (Ptide) metabolism without radiolabeling.