A method was developed for the quantitative isolation of tissue sphingomyelins.Total lipid extracts of tissues were subjected to selective hydrolysis to destroy ester phosphatides and plasmalogens. The water-soluble hydrolysis products were removed by solvent fractionation. The sphingomyelins were separated from other lipids on silicic acid columns.Sphingomyelins were prepared from beef heart, lung, kidney, adrenal, brain, liver, and adipose tissue. The sphingomyelins were analyzed by thin-layer chromatography, paper chromatography of hydrolysis products, and infrared spectroscopy. Fatty acids were analyzed by gas-liquid chromatography.Methods for preparation and analysis of sphingomyelins are discussed.