Presilphiperfolan-8 beta-alpha synthase, encoded by the BcBOT2 gene from the necrotrophic plant pathogen Botrytis cinerea, catalyzes the multistep cyclization of farnesyl, diphosphate (2) to the tricyclic sesquiterpene alcohol presilphiperfolan-8 beta-alpha (3), the preursor of the phytotoxin botrydial, a strain-dependent fungal virulence factor. Incubation of (1R)-[1-H-2]farnesyl diphosphate (2b) with recombinant presilphiperfolan-8 beta-alpha synthase gave exclusively (5R)-[5 alpha-H-2]-3b, while complementary incubation of (1S)-[1-H-2]FPP (2c) gave (5S)-[5 beta-H-2]-3c. These results established that cyclization of farnesyl diphosphate involves displacement of the diphosphate group from C-1 with net inversion of configuration and ruled out the proposed intermediacy of the cisoid conformer of nerolidyl diphosphate (9) in the cyclization. White not a mandatory intermediate, (3R)-nerolidyl diphosphate was shown to act as a substrate surrogate. Cyclization of [13,13,13-H-2(3)] farnesyl diphosphate (2d) gave [14,14,14-H-2(3)]-3d, thereby establishing that electrophilic attack takes place exclusively on the si face of the 12,13-double bond of 2. The combined results provide a detailed picture of the conformation of enzyme-bound farnesyl diphosphate at the active site of presilphiperfolan-8 beta-ol, synthase.