(2R)-2-amino-6-hydroxy-4-hexynoic acid, (2RS)-2-amino-4,5-hexadienoic acid, (2S)-2-amino-4-hexynoic acid, and 2-amino-5-chloro-5-hexenoic acid were isolated and characterised from the fruit bodies of Amanita miculifera, with the first and the last mentioned amino acids being new natural products. Their structures were based on analysis of the results from elementary analysis, oxidation with KMnO4, determination of optical rotations, 1H NMR-spectra, and hydrogenation over Adams or Lindlar catalysts. (2RS)-2-Amino-6-hydroxy-4-hexynoic acid was synthesized and optically resolved by renal acylase to confirm its natural (2R)configuration. The configuration at C-2 of 2-amino-4,5-hexadienoic acid was also assumed from its ORD spectrum, as well as that of its hydrogenation product. The optical rotation (2-amino-5-chloro-5-hexenoic acid) was not determined because it was isolated in trace amounts; it was also prepared from 2-amino-5hexynoic acid, although in very low yield. A possible biosynthetic route leading to the non-protein amino acids in this fungus is presented.