Tobacco epiaristolochene and hyoscyamus premnaspirodiene synthases (TEAS and HPS) catalyze the cyclizations and rearrangements of (E,E)-farnesyl diphosphate (FPP) to the corresponding bicyclic sesquiterpene hydrocarbons. The complex mechanism proceeds through a tightly bound (R)-germacrene A intermediate and involves partitioning of a common eudesm-5-yl carbocation either by angular methyl migration, or by C-9 methylene rearrangement, to form the respective eremophilane and spirovetivane structures. In this work, the stereochemistry and timing of the proton addition and elimination steps in the mechanism were investigated by synthesis of substrates bearing deuterium labels in one or both terminal methyl groups, and in the pro-S and pro-R methylene hydrogens at C-8. Incubations of the labeled FPPs with recombinant TEAS and HPS, and with the chimeric CH₄ hybrid cyclase having catalytic activities of both TEAS and HPS, and of unlabeled FPP in D₂O, together with gas chromatography–mass spectrometry (GC–MS) and/or NMR analyses of the labeled products gave the following results: (1) stereospecific CH₃ → CH₂ eliminations at the cis-terminal methyl in all cases; (2) similar primary kinetic isotope effects (KIE) of 4.25–4.64 for the CH₃ → CH₂ eliminations; (3) a significant intermolecular KIE (1.33 ± 0.03) in competitive cyclizations of unlabeled FPP and FPP-d₆ to premnaspirodiene by HPS; (4) stereoselective incorporation of label from D₂O into the 1β position of epiaristolochene; (5) stereoselective eliminations of the 1β and 9β protons in formation of epiaristolochene and its Δ¹⁽¹⁰⁾ isomer epieremophilene by TEAS and CH₄; and (6) predominant loss of the 1α proton in forming the cyclohexene double bond of premnaspirodiene by HPS and CH₄. The results are explained by consideration of the conformations of individual intermediates, and by imposing the requirement of stereoelectronically favorable proton additions and eliminations.