SUMMARY A system of two-dimensional thin-layer chromatography was developed that separated rat liver phosphatides into several phosphate-positive spots in about 2 hr developing time. Characteristic hydrolysis products derived from phosphatidyl serine, phosphatidyl ethanolamine, phosphatidyl inositol, phosphatidyl choline, sphingomyelin, and lysophosphatidyl choline were identified. The hydrolytic products of “phosphatidic acid” were not definitely characterized. The application of thin-layer chromatography as described for rat liver phosphatides can be extended to phosphatide extracts of other tissues. The trend toward widespread use of thin-layer chromatography (TLC) was accelerated by the work of Stahl (1-3) who demonstrated its potential usefulness in lipid research. In an excellent review, Mangold (4) presented a detailed description of commercially available apparatus and several techniques used in TLC as well as applications of the method to lipids in general. One-dimensional TLC has been applied to the separation of phosphatides from brain (5, 6), serum (7-g), and other sources (10) with varying degrees of success. It was observed in this laboratory that one-dimensional TLC did not separate all of the phosphatides found in a rat liver extract with the solvent systems used. Therefore, a system of two-dimensional TLC was applied. Tentative identification of the phosphatides was made possible by the application of different color-developing reagents. Further identification was accomplished by eluting the phosphatides, hydrolyzing them, and identifying the hydrolytic cleavage products with standards by TLC. In this way, the seven phosphatide spots were identified as “phosphatidic acid” (PM), phosphatidyl serine (PhS) , phosphatidyl ethanolamine (PhE), phosphatidyl inositol (PhI), phosphatidyl choline (PhC) , sphingomyelin (Sph), and lysophosphatidyl choline (L-phc).