Neutral tryptophan metabolites accompanying echinuline in Aspergillus spp. have recently received structural, biosynthetic, and synthetic interest. This study reports the spectroscopic evidences (including 1H-NMR, 13C-NMR, and mass spectroscopy) that allow assigning structural formula (3) to cryptoechinuline G, C29H35N3O2, an amorphous solid isolated from the fungal mat of Aspergillus ruber grown on sugar beet molasses. The preferential choice of formula (3) over isomer (4) is based on the following considerations: a) In the 13C-NMR spectrum (d6-DMSO), the signals at δ 123 and 109.5 (doublets) for indole carbons 6 and 7 fall in a region typical of unsubstituted positions (whereas metabolites with substituents at 6 and 7 show resonances at ~δ 134 and 123, respectively); b) The carbon at position 3 absorbs at δ 101, a shift possibly due to the shielding γ-effect of the CH2 at position 4a (compared to δ 103-104 in similar metabolites without a 4-substituent); c) The temperature-dependent 1H-NMR behavior of one CH2 group (line width variation, splitting at low temperatures, coalescence at 15°C) is attributed to restricted conformational mobility of the isopentenyl chain at position 4 of (3), as supported by steric models. Cryptoechinuline G is chemotaxonomically novel among isoprenylated tryptophan metabolites related to echinuline due to the presence of two adjacent isopentenyl chains, one at position 4 (typical of ergot alkaloids and cyclopiasonic acid). Feeding experiments with 3H-labelled neoechinuline A and B, and isoechinuline A (2), designed to clarify the metabolic significance of tryptophan's α,β-desaturation relative to the timing of indole nucleus isoprenylation in the biosynthesis of (3), are ongoing.