A method is described for the separation and analysis of phospholipids from rat-liver nuclear envelope and endoplasmic reticulum. The procedure employs a liquid environment, to which antioxidants can be added, and results in separation of NL, PE, PI, PS, and PC in 99% purity in 12 min; analytical columns and a radial compression system may be employed. The procedure results in phospholipids with a large proportion of highly unsaturated fatty acids; some differences in fatty acid distributions were found when nuclear envelope phospholipid fractions were compared with the corresponding fractions from endoplasmic reticulum.