The binding of polyphosphoinositides (phosphatidylinositol phosphate and phosphatidylinositol bisphosphate) to the antibiotic neomycin is utilized for the purification of these lipids. Neomycin is reductively coupled to reactive glass beads (Glycophase-CPG) and serves as the stationary phase in column chromatography. A total lipid extract is prepared from tissues with chloroform-methanol-KC1 or chloroform-methanol-HC1 and washed once with acidified methanol-water. After the addition of an equal volume of methanolic 200 mM ammonium acetate, the extract is directly applied to the column. All lipids but the polyphosphoinositides are removed from the column by rinsing with 150 mM ammonium acetate in chloroform-methanol-water. Increasing the salt concentration to 600 mM elutes phosphatidylinositol phosphate. While further increases in ionic strength are not sufficient for a quantitative removal of phosphatidylinositol bisphosphate, the lipid is completely eluted by the addition of either ammonia or HC1 to the solvent. The column can be recycled and used repeatedly.