Ergot alkaloid pathway reconstruction in Aspergillus nidulans is an approach used to better understand the biosynthesis of these mycotoxins. An engineered strain named A. nidulans WFC (expressing ergot alkaloid synthesis genes dmaW, easF, and easC) produced the established intermediate N-methyl-4-dimethylallyltryptophan, as well as an uncharacterized ergot alkaloid. We investigated the chemical structure of the new metabolite and its role in the ergot alkaloid pathway. Mass spectrometry, labeling, and NMR studies showed that the unknown ergot alkaloid, designated here as ergotryptamine, differed from N-methyl-4-dimethylallyltryptophan by the loss of the carboxyl group, addition of a hydroxyl group, and shift in position of a carbon–carbon double bond. Feeding studies with Aspergillus mutants did not show ergotryptamine turnover, suggesting it is a pathway byproduct as opposed to an authentic intermediate. Several Epichloë species also produced this metabolite, and further investigations revealed the equivalency of ergotryptamine with an Epichloë-derived ergot alkaloid provisionally described as 6,7-secolysergine.