Tobacco 5-epi-aristolochene synthase (TEAS) catalyzes the Mg(II)-dependent cyclizations and rearrangements of (E,E)-farnesyl diphosphate (PP) to the bicyclic sesquiterpene hydrocarbon via a tightly bound (+)-germacrene A as a deprotonated intermediate. With the native enzyme, only a few percent of the putative germacrene A intermediate is released from the active site during the catalytic cycle. 6-Fluorofarnesyl PP was designed and synthesized with the aim of arresting the cyclization-rearrangement mechanism en route to 5-epi-aristolochene. Indeed, incubation of (2E,6Z)-6-fluorofarnesyl PP with recombinant TEAS afforded (-)-1-fluorogermacrene A as the sole product in 58% yield. Steady-state kinetic experiments with farnesyl PP and the 6-fluoro analogue showed that the overall catalytic efficiencies (k(cat)/K(m)) are essentially the same for both substrates. 1-Fluorogermacrene A was characterized by chromatographic properties (TLC, GC), MS, optical rotation, UV, IR and (1)H NMR data, and by heat-induced Cope rearrangement to (+)-1-fluoro-beta-elemene. (1)H NMR spectra at room temperature revealed that this (E,E)-configured fluorocyclodecadiene exists in solution as a 7:3 mixture of UU and UD conformers. 1-Fluorogermacrene A underwent trifluoroacetic acid-catalyzed cyclization to give three 1alpha-fluoroselinene isomers at a rate estimated to be about 1000 times slower than that of the similar cyclization of (+)-germacrene A to the parent selinenes.