The principal free amino acid present in seed of Trigonella foenum-graecum has been isolated and identified as (2S, 3R, 4R)-4-hydroxyisoleucine. This compound has not been reported previously as a constituent of higher plants, but it is a component of the toxic peptide, γ-amanitin, produced by Amanita phalloides. The (2S, 3R, 4R)-isomer lactonizes readily under acidic conditions, whilst strong acid causes partial epimerization. The (2R, 3R, 4R)-isomer forms a minor component of Trigonella seed. The 4-hydroxyisoleucine content of fenugreek increases during the growth of seedlings and plants, and ¹⁴C-isoleucine was used effectively as a biosynthetic precursor.