In the course of the investigation of indole metabolites from Aspergillus chevalieri, we reported the isolation and structure of L-alanyl-2-(1,1-dimethylallyl)-L-tryptophan anhydride (I) previously. Recently, we isolated a compound related to the above metabolite from this mold, and this paper describes the structural elucidation of this new indole metabolite. Aspergillus chevalieri (Mangin) Thom et Church IFO 4090 was grown as a surface culture for 3 weeks at 24•Ž on potato extract medium. The culture filtrates (80 liters) were stirred with activated charcoal, the adsorbed metabolites were eluted with acetone, and the acetone extracts (23g) were chromatographed on a silicic acid column. Elution with benzene-acetone (2:1, v/v) gave 650mg of the new metabolite (II). Metabolite II forms colorless needles, mp 290-292•Ž (decomp.), [ƒ¿]25D +36•‹ (c=0.5, AcOH) and +18•‹ (c=0.05, EtOH) when crystallized from methanol-acetone. Its molecular formula C14H15O2N3 was deduced from mass spectrometry [m/e: 257 (M+)] and elemental analysis. The presence of an indole ring system in II was indicated by its UV absorptions at 220, 273, 280 and 290 nm and positive reaction to Ehrlich's reagent. It gave a negative reaction to ninhydrin reagent. Its IR spectrum showed absorption at 3420cm-1 (secondary amine group of indole ring system) and at 1675 and 1665cm-1 (carbonyl absorption of amide groups). Treatment of II with 6 N hydrochloric acid afforded L-alanine and L-tryptophan, which were identical with authentic L-alanine and L-tryptophan by comparison of optical rotations and IR spectra, suggesting a dioxopiperazine ring system formed from L-tryptophan and L-alanine. NMR spectrum analysis of proton signals (alanyl methyl protons, allylic methylene protons, -NH group protons in indole and dioxopiperazine rings, aromatic protons) further supported the structure. The evidence indicated this metabolite is L-alanyl-L-tryptophan anhydride (II), whose properties were identical with those of a synthetic sample.