Previous studies have shown that ricinoleic acid from castor bean oil of Ricinus communis is synthesized by the direct hydroxyl substitution of oleate, while it has been proposed that ricinoleate is formed by hydration of linoleate in the ergot fungus Claviceps purpurea. The mechanism of the enzymes specific to ricinoleate synthesis has not yet been established, but hydroxylation and desaturation of fatty acids in plants apparently involve closely related mechanisms. As mechanistic differences in the enzymes involved in the biosynthesis of natural products can lead to different isotopic distributions in the product, we could expect ricinoleate isolated from castor or ergot oil to show distinct (2)H distribution patterns. To obtain information concerning the substrate and isotope effects that occur during the biosynthesis of ricinoleate, the site-specific natural deuterium distributions in methyl ricinoleate isolated from castor oil and in methyl ricinoleate and methyl linoleate isolated from ergot oils have been measured by quantitative (2)H NMR. First, the deuterium profiles for methyl ricinoleate from the plant and fungus are equivalent. Second, the deuterium profile for methyl linoleate from ergot is incompatible with this chemical species being the precursor of methyl ricinoleate. Hence, it is apparent that 12-hydroxylation in C. purpurea is consistent with the biosynthetic mechanisms proposed for R. communis and is compatible with the general fundamental mechanistic similarities between hydroxylation and desaturation previously proposed for plant fatty acid biosynthesis.