β-Hydroxy-L-valine was isolated from fruiting bodies of Pleurocybella porrigens (Fr.) Sing. In a previous paper [1], we reported the free amino acid contents of fruiting bodies of 113 mushroom species, as determined by automatic amino acid analysis of their 70% ethanol extracts. In the course of the experiment, some of the mushrooms were found to contain several unusual amino acids. This paper describes the isolation and identification of an unknown ninhydrin positive compound, hereafter termed as 1, from the fruiting bodies of the edible mushroom, Pleurocybella porrigens (Fr.) Sing. Compound 1 was detected on the chromatogram of the amino acid analyser (Li-citrate buffer system) and was completely overlapped with threonine. The two were distinguishable by TLC (silica gel and Avicel cellulose). The isolation of 1 was accomplished by chromatography using several ion-exchange resins, Avicel cellulose and silica gel, followed by crystallization. From the elemental analysis and SIMS, the molecular formula of 1 was estimated to be C₅H₁₁NO₃. The ninhydrin reaction of 1 on paper was completely inhibited by Cu²⁺, showing that 1 is a α-monoamino acid. On the ¹H NMR of 1 (in D₂O), one gem-dimethyl group (δ 1.26 and 1.47) and one methyne singlet (δ 3.65), which indicated the β carbon atom to be fully substituted, were observed. The absorption band at 1167 cm⁻¹ in the IR showed the presence of a tertiary hydroxyl group in the molecule [2]. These facts strongly suggested that 1 is β-hydroxyvaline. This was further supported by the formation of valine from the reduction with HI-red P [3], and the detection of glycine in the degradation product by barium hydroxide [4]. Optical rotation measurements on 1 performed in H₂O and 6 M HCl solution showed the shift to more positive rotations in acid associated with an L configuration. The IR of 1 correlated well with that of a synthetic specimen of β-hydroxy-L-valine [5]. Although β-hydroxy-L-valine was first found as a constituent of the antibiotic YA-56 [6, 7], it is thought that the evidence presented here is the first to demonstrate its natural occurrence in a free form.