We studied the effect of metabolic inhibitors on essential oil biosynthesis in terms of change in rate of sodium [2-14C]-acetate (activity 0.1 mCi, specific activity 34.51 mCi mmole-1) incorporation into essential oil in Cymbopogon flexuosus (Ness ex Steud) Wats mutant cv. GRL-1 leaves. Out of seven metabolic inhibitors tested, six severely inhibited the label incorporation into essential oil and in particular with their higher concentration. The order of effectiveness of inhibitors expressed as percent inhibition was as follows: sodium arsenate (74.4%) > miconazole (64.35%) > sodium fluoride and sodium borate (47.0%) > 2, 4-di-nitrophenol (DNP) (24.0%). Essential oil biosynthesis, however, could not be inhibited following the mevastatin treatment. The study revealed that metabolic inhibitors interrupted the supply of precursors and co-factors like NADPH, ATP etc. required for cytosolic acetate-mevalonate pathway of essential oil biosynthesis. However, studies with mevastatin clearly supported that besides acetyl Co-A, photosynthetic metabolites, ATP, NADPH, other carbon sources are also utilized for essential oil biosynthesis.