L-THREO-γ-hydroxycitrulline was isolated and identified from the seeds of Vicia pseudo-orobusta. The structure was clarified from the results of elementary analysis, ¹H NMR spectrum, enzymic deamination and comparison of the hydrolysis product with the authentic threo- and erythro-γ-hydroxyornithine. Previously we reported the isolation and characterization of Nᵟ-benzoyl-L-ornithine and N-benzoyl-L-γ-hydroxyornithine from the seeds of Vicia pseudo-orobusta [1]. Subsequently the configuration of the two asymmetric carbon atoms of the latter amino acid was unequivocally determined as L-threo-form by comparison with the synthetic samples [2,8]. The seeds of V. pseudo-orobusta contain still other ninhydrin-positive substances yielding on hydrolysis γ-hydroxyornithine. One of these proved now to be L-THREO-γ-hydroxycitrulline. By the use of cellulose CC we obtained γ-hydroxycitrulline from the neutral and acidic amino acid fraction. The result of elementary analysis was in good agreement with the formula C₆H₁₃N₃O₄. It gave a brownish violet coloration with ninhydrin and the colour turned to normal violet with time. Ehrlich reagent yielded a yellow colour just as in the case of citrulline. Though on strong alkaline hydrolysis it gave a mixture of threo- and erythro-γ-hydroxyornithine, only the former was detected in the mild alkaline hydrolysis products. Also, strong acid yielded only threo-γ-hydroxyornithine. Epimerization of γ-hydroxyornithine is known to occur more rapidly in alkaline than in acidic solution [2]. Further, ¹H NMR spectrum of the natural γ-hydroxycitrulline was very similar to that of Nᵟ-benzoyl-L-threo-γ-hydroxyornithine and different from that of the erythro-form [1,3]. An experiment with L-amino acid oxidase ascertained that our isolate belongs to the series of L-amino acids. γ-Hydroxycitrulline was first identified as a natural product by Bell and Tirimanna from Vicia faba and V. unijuga but not isolated [4]. The first isolation was carried out by Inatomi et al. from young seeds of Vicia joto [9]. The stereochemical nature, however, was not studied; Synthetic nopaline and isonopaline were separated preparatively by anion exchange chromatography and their configurations were ascertained by an enzymic method. Nopaline prepared from crown gall of Helianthus annuus corresponded to authentic L-,D-form (nopaline). The strains of Agrobacterium tumefaciens which are known to utilize nopaline as sole nitrogen source grew also with this form, but not with L-,L-form (isonopaline).