The biosynthesis of ergot alkaloids commences with the alkylation of tryptophan by dimethylallylpyrophosphate to give 4-dimethylallyltryptophan (Ia). The next step is probably a hydroxylation to give 45Z-4-hydroxy-3-methyl-Δ2-butenyl-tryptophan (Ib), a compound of undefined stereochemistry isolated from Claviceps purpurea cultures. We report the isolation of N-methyl-4-dimethylallyltryptophan (II) from oxygen-deprived Claviceps fusiformis cultures. Claviceps fusiformis was aerobically grown in submerged shaken flasks and stirred fermenters; anaerobic conditions were imposed upon alkaloid production onset, followed by three more days of culture. Clavine alkaloids were extracted with chloroform at alkaline pH, and amphoteric metabolites with n-butanol at neutral pH. The butanol extract—containing chanoclavines and other oxygenated clavine alkaloids—was chromatographed on silica using chloroform/methanol/ammonia as eluant. Small amounts of clavicipitic acid isomers, N-methyl-4-dimethylallyltryptophan (II), and 4-dimethylallyltryptophan (Ia) were obtained. N-methyl-4-dimethylallyltryptophan crystallized from methanol as needles (m.p. 232°C) with λmax 274, 280, 295 nm; vmax 3580, 3250 (broad), 1640, 1400, 770 cm⁻¹; NMR (CD₃COOD) δ 8.64 (s, 6H), 7.64 (s, 3H), 5.06 (t, 1H, J 7.0Hz), τ 6.3–7 (complex, 4H); m/e 286, 198 (100%), 156, 155, 154. Its structure was supported by: identical allylic methyl chemical shifts to bissecodehydrocyclopiazonic acid (III); electron impact fragmentation (allylic cleavage yielding m/e 198, followed by cyclization to tricyclic ions m/e 156, 155, 154 via C-3 unit elimination—possible only if side chains are at indole positions 3 and 4); mass spectrum of hydrogenation product (IV) (m/e 288, 200, 144 (100%)) with a metastable peak (m/e 103.6) indicating m/e 144 arises from m/e 200 via (CH₃)₂CH=CH₂ loss (analogous to cyclopiazonic acid series). ¹⁴C-methyl-methionine feeding experiments showed (II) was the only labeled amphoteric tryptophan metabolite (4–8% incorporation); refeeding labeled (II) yielded labeled agroclavine (V) (1.4% incorporation), with other clavine alkaloids also labeled but unpurified. Whether (II) is an obligatory ergot alkaloid precursor or an oxygen deprivation-induced accumulation product is unestablished; (II) is undetectable in normal aerobic cultures. However, its facile production suggests the N-methylation step—whether preceding or following 4-dimethylallyl substituent Z-methyl group hydroxylation—occurs before decarboxylation/cyclization to tricyclic chanoclavines (VI).