The biosynthesis of penitrem A has been studied with both ¹³C- and ²H-labelled precursors, viz. [1-¹³C]-, [2-¹³C]-, [1,2-¹³C₂]-, and [1-¹³C,2-²H₃]-acetate, [2-¹³C]-, [2,3-¹³C₂]-, [2-²H₂]- and [5-²H₂]-mevalonate. The results show that penitrem A is derived from tryptophan, which contributes the indole moiety of the metabolite, geranylgeranylpyrophosphate, and two isopentenylpyrophosphate units. A 1,2-bond migration, involving the 2,3-bond of a mevalonate unit, occurs in the course of the biosynthesis and results in the observation of a one-bond (C,C) coupling between two [1-¹³C]acetate-derived carbon atoms and between two [2-¹³C]acetate-derived carbon atoms. Analysis of the one-bond (C,C)coupling constants in [2-¹³C]acetate-derived penitrem A showed that [1,2-¹³C₂]acetate was formed during the fermentation. Although loss of water from an hydroxyisopropyl group to form the isopropenyl function present in penitrem A should proceed with retention of the stereochemical integrity of the two methyl groups, isomerization of the double bond causes equal distribution of ¹³C label between C-36 and C-38 and precludes any stereochemical deductions.