Cassava (Manihot esculenta Crantz) is an important starch-rich crop, but the storage roots only have a short shelf-life due to post-harvest physiological deterioration (PPD), which includes the over-production and polymerisation of hydroxycoumarins. Key aspects of coumarin secondary-metabolite biosynthesis remain unresolved. Here we exploit the accumulation of hydroxycoumarins to test alternative pathways for their biosynthesis. Using isotopically labelled intermediates (p-coumarate-2-(13)C, caffeate-2-(13)C, ferulate-2-(13)C, umbelliferone-2-(18)O and esculetin-2-(18)O), we show that the major biosynthetic pathway to scopoletin and its glucoside, scopolin, in cassava roots during PPD is through p-coumaric, caffeic and then ferulic acids. An alternate pathway through 2',4'-dihydroxycinnamate and umbelliferone leads to esculetin and esculin. We have used C(18)O(2)-carboxylate-labelled cinnamic and ferulic acids, and feeding experiments under an atmosphere of (18)O(2), to investigate the o-hydroxylation and cyclisation steps. We demonstrate that the major pathway is through o-hydroxylation and not via a proposed spirolactone-dienone intermediate.