Sesquiterpene synthases are a family of enzymes that catalyze farnesyl pyrophosphate (FPP) cyclization via alternative pathways to produce a variety of cyclic sesquiterpene products. Catalysis by several of these enzymes, including tobacco 5-epi-aristolochene synthase (TEAS), has been proposed to include the formation of germacrene A as a stable intermediate. Neither germacrene A nor any other intermediate is released from sesquiterpene synthase active sites during normal catalysis. Evidence to support the intermediacy of germacrene A has been derived from investigations of aristolochene synthases from Aspergillus terreus and Penicillium roquefortii (Cane, D. E. Chem. Rev. 1990, 90, 1089-1103 and references therein. Cane, D. E.; Bryant, C. J. Am. Chem. Sec. 1994, 116, 12063-12064. Cane, D. E.; Tsantrizos, Y. S. J. Am. Chem. Soc. 1996, 118, 10037-10040). However, until the present investigations of TEAS, formation of this postulated intermediate has never been directly demonstrated. TEAS catalyzes the cyclization of FPP to 5-epi-aristolochene, a precursor of a tobacco phytoalexin, capsidiol. Based upon the three-dimensional structure of TEAS, a detailed mechanism has been proposed for TEAS catalysis that includes the prediction that proton donation by Y520 is responsible for the activation of germacrene A to a eudesmane cation (Starks, C. M.; Back, K.; Chappell, J.; Noel, J.P. Science 1997, 277, 1815-1820). In the present investigation, a Y520F point mutation is introduced into TEAS (TEAS-Y520F) by site-directed mutagenesis. In the presence of H-3-FPP, TEAS-Y520F produces hexanes-extractable H-3 With a catalytic efficiency approximately 3% that of nonmutated, recombinant TEAS. The hexanes-extractable 3H is identified as germacrene A, mit 204, through direct CC-MS comparison to an authentic sample. This observation confirms the intermediacy of germacrene A in TEAS catalysis, supports the postulated production of germacrene A by a variety of other sesquiterpene synthases, and also confirms the proposed role of Y520 in TEAS catalysis.