During radioisotopic investigations of receptor-mediated breakdown of triphosphoinositide to inosito triphosphate and diglyceride (DG) and conversion of the latter compound to monoglyceride (MG) and fatty acid (FA), a thin-layer chromatographic (TLC) system was needed that would ensure that FA, MG and DG would be sufficiently separated from each other and other neutral lipids to allow them to be scraped from the plate and counted. Of the existing chromatographic systems for the separation of neutral lipidslw6, only the systems of Freeman and West' and Chabard et aL3 seemed to provide this degree of separation. The latter system, however, requires specialized equipment3 and the separation reported by Freeman and West6 could not be reproduced as FA overlapped DG. Studies were therefore undertaken to design a system that would provide the required separation. The resulting system effects a separation that allows the following lipid fractions listed in order of increasing RF values, to be scraped from the plate for determination: phospholipids (PL, at the origin), FA, MG, cholesterol (C), DG, triglyceride (TG) and cholesterol esters (CE). The system described here was compared with that of Freeman and West6, which provided guidelines in designing the present chromatographic system.