The 13C n.m.r. spectra of aphidicolin and its derivatives have been assigned. The enrichment and coupling patterns of aphidicolin biosynthesized from [1-13C]-, [2-13C]-, and [1,2-13C2]-acetate were determined and used to define the constituent isoprene units. The numbers of 2- and 5-mevalonoid hydrogen atoms incorporated into aphidicolin were determined by 3H labelling. The generation of2H–13C coupling which was observed in the 2H n.m.r. spectrum of aphidicolin biosynthesized from [4-2H2,3-13C] mevalonic acid, has established the migration of a 9β-hydrogen atom to C-8 during the biosynthesis.