Artemisinin from Artemisia annua has become one of the most important drugs for malaria therapy. Its biosynthesis proceeds via amorpha-4,11-diene, but it is Still unknown whether the isoprenoid precursors units are obtained by the mevalonate pathway or the more recently discovered non-mevalonate pathway. In order to address that question, a plant of A. annua was grown in at) atmosphere containing 700 ppM Of (CO2)-C-13 for 100 min. Following a chase period of 10 days, artemisinin was isolated and analyzed by C-13 NMR spectroscopy. The isotopologue pattern shows that artemisinin was predominantly biosynthesized from (E,E)-farnesyl diphosphate (FPP) whose central isoprenoid unit had been obtained via the non-mevalonate pathway. The isotopologue data confirm the previously proposed mechanisms for the cyclization of (E,E)-FPP to amorphadiene and its oxidative conversion to artemisinin. They also support deprotonation of a terminal allyl cation intermediate as the final step in the enzymatic conversion of FPP to amorphadiene and show that either of the two methyl groups can undergo deprotonation. (C) 2009 Elsevier Ltd. All rights reserved.