A unidimensional thin-layer chromatography system for the separation of phospholipids has been developed. The method employs precoated plates impregnated with boric acid and is capable of resolving eight phospholipid standards, including phosphatidylinositol, phosphatidylserine, and phosphatidylglycerol. Liver, kidney, and platelet phospholipids of the rat were resolved into six major classes using this method. Each was judged to be pure by two-dimensional thin-layer chromatography. The use of boric acid thin-layer chromatography for the separation of phospholipids should find wide application.