Abstract The three pinene synthases (cyclases) from common sage ( Salvia officinalis) catalyze the conversion of geranyl pyrophosphate to the bicyclic olefins (+)-α-pinene and (+)-camphene (cyclase I), (−)-α-pinene, (−)-β-pinene, and (−)-camphene (cyclase II), and (+)-α-pinene and (+)-β-pinene (cyclase III), in addition to smaller amounts of monocyclic and acyclic monoterpene olefins. [1- 3H,4- 2H 2]- and [10- 2H 2]-geranyl pyrophosphates were prepared and used in conjunction with 1- 3H- and 1- 3H,10- 2H 3-labeled geranyl precursors to examine the isotope effects attending the C4- and C10-deprotonation steps in the enzymatic synthesis of the pinenes. The observation of isotopically sensitive branching within each set of stereochemically related bicyclic olefins confirmed that each product set was synthesized by the respective pinene synthase by partitioning of common carbocationic intermediates along different reaction channels at the active site. The changes in product distribution resulting from deuterium substitution at C4 and C10 of the substrate were used to determine kinetic isotope effects (KIEs) for the terminating deprotonations; these observed KIEs represent the lower limits of the intrinsic isotope effects. The intramolecular isotope effects for the methyl-methylene elimination in β-pinene formation by cyclases II and III were also evaluated with [10- 2H 2]geranyl pyrophosphate as substrate and by MS analysis of the olefin products. The intramolecular KIEs ( k H/ k D = 3.0 and 3.5) were significantly higher than the observed KIEs determined from product ratios ( k H/ k D = 1.7 and 2.6) since the former involves considerably less masking of the intrinsic isotope effects.