The natural OLE-1 high-oleic castor mutant has been characterized, demonstrating that point mutations in the FAH12 gene are responsible for the high-oleic phenotype. The contribution of each mutation was evaluated by heterologous expression in yeast, and lipid studies in developing OLE-1 seeds provided new evidence of unusual fatty acids channeling into TAGs. Ricinus communis L. is a plant of the Euphorbiaceae family well known for producing seeds whose oil has a very high ricinoleic (12-hydroxyoctadecenoic) acid content. Castor oil is considered the only commercially renewable source of hydroxylated fatty acids, which have many applications as chemical reactants. Accordingly, there has been great interest in the field of plant lipid biotechnology to define how ricinoleic acid is synthesized, which could also provide information that might serve to increase the content of other unusual fatty acids in oil crops. Accordingly, we set out to study the biochemistry of castor oil synthesis by characterizing a natural castor bean mutant deficient in ricinoleic acid synthesis (OLE-1). This mutant accumulates high levels of oleic acid and displays remarkable alterations in its seed lipid composition. To identify enzymes that are critical for this phenotype in castor oil, we cloned and sequenced the oleate desaturase (FAD2) and hydroxylase (FAH12) genes from wild-type and OLE-1 castor bean plants and analyzed their expression in different tissues. Heterologous expression in yeast confirmed that three modifications to the OLE-1 FAH12 protein were responsible for its weaker hydroxylase activity. In addition, we studied the expression of the genes involved in this biosynthetic pathway at different developmental stages, as well as that of other genes involved in lipid biosynthesis, both in wild-type and mutant seeds.