Two to three days after harvesting, cassava (Manihot esculenta Crantz) roots suffer from post-harvest physiological deterioration (PPD) when secondary metabolites are accumulated. Amongst these are hydroxycoumarins (e.g. scopoletin and its glucoside scopolin) which play roles in plant defence and have pharmacological activities. Some steps in the biosynthesis of these molecules are still unknown in cassava and in other plants. We exploit the accumulation of these coumarins during PPD to investigate the E-Z-isomerisation step in their biosynthesis. Feeding cubed cassava roots with E-cinnamic- 3,20,30,40,50,60-d5 acid gave scopoletin-d2. However, feeding with E-cinnamic-3,20,30,40,50,60-d6 and E-cinnamic-2,3,20,30,40,50,60-d7 acids, both gave scopoletin-d3, the latter not affording the expected scopoletin- d4. We therefore synthesised and fed with E-cinnamic-2-d1 when unlabelled scopoletin was biosynthesised. Solely the hydrogen (or deuterium) at C2 of cinnamic acid is exchanged in the biosynthesis of hydroxycoumarins. If the mechanism of E-Z-cinnamic acid isomerisation were photochemical, we would not expect to see the loss of deuterium which we observed. Therefore, a possible mechanism is an enzyme catalysed 1,4-Michael addition, followed by r-bond rotation and hydrogen (or deuterium) elimination to yield the Z-isomer. Feeding the roots under light and dark conditions with E-cinnamic- 2,3,20,30,40,50,60-d7 acid gave scopoletin-d3 with no significant difference in the yields. We conclude that the E-Z-isomerisation stage in the biosynthesis of scopoletin and scopolin, in cassava roots during PPD, is not photochemical, but could be catalysed by an isomerase which is independent of light.