The lipid composition of the mycobint and photobiont symbiotic partners of lichenized ascomycetes varies greatly. The aim of this study was to compare the profile of the major sterols in two closely related lichens from the genus Lobaria with different photobionts. The three-component lichen Lobaria pulmonaria has two photobionts. While the main photobiont is the chlorophycean alga Symbiochloris reticulata, this lichen contains small amounts of the cyanobacterium Nostoc. By contrast, the cyanobacterium Nostoc is the main photobiont in Lobaria retigera. Relatively loosely bound sterols were extracted using a chloroform–methanol mixture, and subsequently, more tightly bound sterols by alkaline saponification. The initial chloroform–methanol extraction step indicated that ergosterol is the principal sterol in both species, with phytosterols constituting a minor fraction. However, the addition of an alkaline saponification step to the standard protocol of sterol extraction greatly increases the release of tightly bound phytosterols, such as campesterol, stigmasterol, and β-sitosterol from L. pulmonaria, but not from L. retigera. Therefore, the mycobionts and Nostoc mainly possess sterols extractable by the standard mixture of chloroform/methanol, while the chlorophycean algal photobiont contains tightly bound sterols. This observation could be important when studying the roles of sterols in the stress tolerance of lichens. Keywords: lichen; Lobaria pulmonaria; Lobaria retigera; photobiont; mycobiont; sterols; saponification.