ABSTRACT Sheep, bovine, mouse, dog and human thyroids incorporate 35–55% (41.5%, 45.9%, 34.4%, 55.4% and 41.1%, respectively) of total lipid radiodine in an unidentified polar thyrolipid fraction moving between phosphatidyl ethanolamine and phosphatidic acid on silica gel G thin layer chromatography in a solvent system CHCl3:MeOH:H2O (80:25:3). This unidentified lipid fraction (Fr. II) is ninhydrin negative, phosphorus negative, alkali labile, acid stable, phospholiphase A stable and orcinol positive indicating the absence of a free amino group, phosphorus group, vinyl ether linkage, vicinal phosphorus ester linkage and the presence of a sugar moiety. Iodination of fraction II seems to be specific for the thyroid gland only, as the other iodide concentrating tissues such as the submaxillary salivary gland and stomach localise most of the radioiodine with phosphatidyl choline and lyso-phosphatidyl ethanolamine fraction respectively. Biologically inactive thyroid tissue or non-enzymic iodination of isolated thyrolipids indicate phosphatidyl choline and/or phosphatidyl ethanolamine as major iodinated lipid fractions. The possible role of fraction II in iodination for homornogenesis in thyroid tissue is discussed.