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- 13C2h [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.