In a previous communication, we reported on the isolation and physicochemical and biological properties of arphamenines A and B, new aminopeptidase B inhibitors which enhance immune responses. This communication reports the structure determination of arphamenines. Arphamenine A (I-a) was obtained as its monohydrochloride with the molecular formula C₁₇H₂₇N₃O₃•HCl, and its structure was determined via elemental analysis, field desorption mass spectrometry, UV, ¹H NMR, and ¹³C NMR spectra, then confirmed by mass spectrometry of its derivative (IV-a) as 5-amino-8-guanidino-4-oxo-2-phenylmethyl octanoic acid. Arphamenine B (I-b), as its monohydrochloride monohydrate with the molecular formula C₁₇H₂₇N₃O₄•HCl•H₂O (containing one more oxygen atom than I-a), was suggested to be p-hydroxy arphamenine A based on spectral data and confirmed by mass spectrometry of its derivative (IV-b) as 5-amino-8-guanidino-2-(4-hydroxyphenylmethyl)-4-oxooctanoic acid. The absolute configuration at C-2 and C-5 was determined by X-ray crystallographic analysis of N'-acetylarphamenine A methyl ester hydrochloride, revealing (2R,5S) configuration, further confirmed by anomalous dispersion and least-squares refinement.