The biosynthesis of (2 S)-2-methyl-2-(4′-methyl-3′-pentenyl)-8-(3″-methyl-2-butenyl)-2 H-1-benzopyran-6-carboxylic acid (gaudichaudianic acid), the major metabolite in leaves and roots of Piper gaudichaudianum Kunth (Piperaceae), has been investigated employing [1- 13C]- d-glucose as precursor. The labelling pattern in the isolated gaudichaudianic acid was determined by quantitative 13C NMR spectroscopy analysis and was consistent with involvement of both mevalonic acid and 2- C-methyl- d-erythritol-4-phosphate pathways in the formation of the dimethylallyl- and geranyl-derived moieties. The results confirmed that both plastidic and cytoplasmic pathways are able to provide isopentenyl diphosphate units for prenylation of p-hydroxybenzoic acid.