A method for the quantification of membrane lipids by acetylation with [3H]acetic anhydride is described. A standard lipid, labelled with 14C or 32P, is added to the sample and is simultaneously acetylated. The 3H/14C or 3H/32P ratios obtained in the acetylated lipids are proportional to the initial lipid concentration. The method has been used to quantify cholesterol, phosphatidylethanolamine, phosphatidylinositol, sphingomyelin, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate in amounts between 2 and 10 nmol.