AbstractFeeding experiments with C14‐labeled and unlabeled unsaturated fatty acids have been used to study the possible routes of formation of the C20‐ and C22‐polyenoic fatty acids of rat liver phosphatides. The acids of the palmitoleate, oleate, linoleate, and linolenate types (considered on the basis of the position of the double bond closest to the methyl end) are apparently formed from the C16 and C18 unsaturated acids of the corresponding types. The results rule out possible transformations of the C20‐ and C22‐polyenoic acids from one type to another, and demonstrate the exclusive introduction of new double bonds toward the carboxyl group. Isomers of linoleate or linolenate in which the double bonds were shifted by one carbon atom toward the carboxyl or methyl groups were incorporated into the phosphatides only to a negligible extent in the form of polyenoic acids.