AbstractThe methyl esters of 9S,10S,13R‐trihydroxy‐11E‐octadecenoic acid, 9S,10R,13S‐trihydroxy‐11E‐octadecenoic acid, and 9S,10R,13R‐trihydroxy‐11E‐octadecenoic acid were prepared from 9S‐hydroperoxy‐10E,12Z‐octadecadienoic acidvia the epoxy alcohols methyl 10R,11R‐epoxy‐9S‐hydroxy‐12Z‐octadecenoate and methyl 10S,11S‐epoxy‐9S‐hydroxy‐12Z‐octadecenoate. The trihydroxyesters, as well as four stereoisomeric methyl 9,12,13‐trihydroxy‐10E‐octadecenoates earlier prepared [Hamberg, M.,Chem. Phys. Lipids 43, 55–67 (1987)], were characterized by thin‐layer chromatography, gas‐liquid chromatography, mass spectrometry, and by chemical degradation. Availability of these chemically defined trihydroxyoctadecenoates made it possible to design a method for regio‐ and stereochemical analysis of 9,10,13‐ and 9,12,13‐trihydroxyoctadecenoic acids obtained from various sources. Application of the method revealed that the mixture of 9,10,13‐ and 9,12,13‐trihydroxyoctadecenoic acids formed during autoxidation of linoleic acid in aqueous medium contained comparable amounts of the sixteen possible regio‐ and stereoisomers. Furthermore, hydrolysis of the allylic epoxy alcohol, methyl 9S,10R‐epoxy‐13S‐hydroxy‐11E‐octadecenoate, yielded a major trihydroxyoctadecenoate,i.e., methyl 9S,10S,13S‐trihydroxyl‐11E‐octadecenoate, together with smaller amounts of methyl 9S,10R,13S‐trihydroxy‐11E‐octadecenoate, methyl 9S,12R,13S‐trihydroxy‐10E‐octadecenoate, and methyl 9S,12S,13S‐trihydroxy‐10E‐octadecenoate.