3-Acetyldeoxynivalenol is the major trichothecene produced by the fungus Fusarium culmorum. The first proven tricyclic intermediate in the biosynthesis of 3-acetyldeoxynivalenol has been shown by in vivo studies to be isotrichodermin, a natural metabolite of F. culmorum. Indeed, the feeding of ring-deuterated isotrichodermin resulted in ring-deuterated 3-acetyldeoxynivalenol as shown by NMR studies. In this work, we have shown that the 3-acetyl group of isotrichodermin is mostly lost in its metabolism to 3-acetyldeoxynivalenol. We have shown by two different approaches that the deacetylation occurs at an early step after the first oxygenation step at C-15. Derivatives of isotrichodermin lacking the 3-acetyl such as 3-deacetyl isotrichodermin or 3-oxo-12,13-epoxytrichothec-9-ene are not precursors to 3-acetyldeoxynivalenol. The role of this acetyl exchange mechanism is not clear presently.