In the preceding paper, the isolation and physicochemical properties of bestatin, a new aminopeptidase B inhibitor produced by an actinomycetes, were described. In this communication, we report the structure determination of bestatin which is [(2S, 3R)-3-amino-2-hydroxy-4-phenylbutanoyl]-L-leucine. Bestatin has the molecular formula C₁₆H₂₄N₂O₄ (M.W. 308) as established by elemental analysis and mass spectrometry. Acid hydrolysis yields L-leucine and a new amino acid (I), which was identified as (2S, 3R)-3-amino-2-hydroxy-4-phenylbutanoic acid via physicochemical analyses including PMR, mass spectrometry, and X-ray crystallographic analysis of its methyl ester hydrobromide. The absolute configuration of I was determined to be 2S, 3R through chemical oxidation of its N-acetyl derivative, PMR analysis of oxazolidone derivatives (distinguishing threo/erythro isomers), and X-ray crystallography. Mass spectrometric analysis of bestatin methyl ester confirmed the amino acid sequence, leading to the conclusion that the structure of bestatin is [(2S, 3R)-3-amino-2-hydroxy-4-phenylbutanoyl]-L-leucine.