A method is presented by which liposomal populations can be separated based on their content of phosphatidylinositol 4,5-bisphosphate (PIP2). Neomycin is reductively coupled to sepharose 2B and used as the stationary phase in column chromatography. Liposomes of egg lecithin (EL) plus acidic phospholipid containing as little as 10 mol% PIP2 are quantitatively retained in 0.2 M NaCl, while liposomes containing other anionic lipids are recovered to 70-97%. It is possible that lipid redistribution of the bilayers upon contact with the sepharose:neomycin adduct may result in the formatin of multiple populations, and the percent retention by the column for each of the EL:phosphoinositide liposomes would be determined by the new equilibrium established as a result of these interactions. When mixed liposomal populations were chromatographed, the recovery in the 0.2 M NaCl eluates was the sum of the individual recoveries, and there is no significant interchange of lipids between the two liposome populations. It is suggested that this procedure may be useful in the preparation of asymmetric liposomes by facilitating separation of populations devoid of PIP2 in the outer leaflet from unreacted liposomes after enzymatic treatment of symmetric PIP2-containing liposomes.