Cyclization of farnesyl diphosphate into amorpha-4,11-diene by amorpha-4,11-diene synthase (ADS) initiates biosynthesis of artemisinin, a clinically important antimalarial drug precursor. Three possible ring-closure mechanisms, two involving a bisabolyl carbocation intermediate followed by either a 1,3-hydride shift or two successive 1,2-shifts, and one involving a germacrenyl carbocation, were proposed and tested by analyzing the fate of farnesyl diphosphate H-1 hydrogen atoms through (1)H and (2)H NMR spectroscopy. Migration of one deuterium atom of [1,1-(2)H(2)]farnesyl diphosphate to H-10 of amorpha-4,11-diene singled out the bisabolyl carbocation mechanism with a 1,3-hydride shift. Further confirmation was obtained through enzyme reactions with (1R)- and (1S)-[1-(2)H]farnesyl diphosphate. Results showed that deuterium of the 1R compound remained at H-6, whereas that of the 1S compound migrated to H-10 of amorpha-4,11-diene. Incorporation of one deuterium into amorphadiene in the cyclization process was observed when the reaction was performed in (2)H(2)O, as evidenced by an increase of 1 amu in the mass of the molecular ion.