The structure of a novel indole alkaloid, glyantrypine 2, a main secondary metabolite of a Yugoslavian isolate of Aspergillus clavatus (IMI 349510), has been elucidated by biosynthetic evidence, mass spectrometry and ¹H and ¹³C NMR techniques. Isolates of A. clavatus otherwise produce the non-benzodiazepine metabolites tryptoquivaline 3 and tryptoquivalone 4, which also contain anthranilic acid and tryptophan residues. Glyantrypine 2 was identified during further screening of fungi for anthranilic acid-containing metabolites using a [carboxyl-¹⁴C]anthranilic acid probe (which detects benzodiazepines like Penicillium aurantiogriseum’s auranthine 1) and designated for its biosynthetic precursors. The compound extends the variety of metabolites biosynthesised from anthranilic acid and tryptophan without forming a benzodiazepine ring (unlike benzodiazepines, which arise from anthranilic acid and α-amino acid condensation). Biosynthetic experiments showed significant incorporation of radiolabel into 2 from [carboxyl-¹⁴C]anthranilic acid (0.05%), [methylene-¹³C]tryptophan (0.07%) and [1-¹⁴C]glycine (0.14%), confirming its composition from these three precursors. Electron impact mass spectrometry revealed a prominent indole fragment ion at m/z 130, an anthranilic acid-glycine moiety at m/z 174, and other fragments (m/z 215, 186, 160) consistent with 2 being formed by condensing anthranilic acid, tryptophan and glycine with the loss of three water molecules and no seven-membered benzodiazepine ring. The ¹H NMR spectrum of 2 in [²H₆]dimethyl sulfoxide ([²H₆]DMSO) showed nine aromatic protons, a broad NH doublet (δ 3.79, dd) coupled to a methylene system, a three-spin system (δ 5.27, t; δ 3.32, dd; δ 3.42, q) identical to tryptophan’s α and β protons, and an indolic NH signal at δ 10.93. Proton-proton correlation spectra confirmed CH-CH₂ and NH-CH' three-spin systems. Connectivity between aromatic protons was established via two-dimensional COSY spectra and analogy with model compounds (e.g., benzodiazepine 5 from a Yugoslavian P. aurantiogriseum isolate, tryptophan, asperlicin 6, fructigenine A). Molecular modelling using MOPAC (version 5.2) with the PM3 Hamiltonian yielded an optimised geometry (dihedral angles of 15° and 104° between C-11 protons and adjacent amide NH proton) consistent with observed couplings. ¹³C NMR spectroscopy in [²H₆]DMSO—supported by DEPT experiments and comparison to model compounds—identified eight quaternary carbons, eleven CH groups and two CH₂ groups, enabling resonance assignments (Table 1) that supported the structure of 2.