Site-saturated mutagenesis experiments were carried out on the His234 residue of Saccharomyces cerevisiae oxidosqualene-lanosterol cyclase (ERG7) to characterize its functional role in ERG7 activity and to determine its effect on the oxidosqualene cyclization/rearrangement reaction. Two novel intermediates, (13aH)-isomalabarica-14(26),17E,21-trien-3β-ol and protosta-20,24-dien- 3β-ol, isolated from ERG7H234X mutants, provided direct mechanistic evidence for formation of the chair-boat 6-6-5 tricyclic Markovnikov cation and protosteryl cation that were assigned provisionally to the ERG7-catalyzed biosynthetic pathway. In addition, we obtained mutants that showed a complete change in product specificity from lanosterol formation to either protosta-12,24-dien-3β-ol or parkeol production. Finally, the repeated observation of multiple abortive and/or alternative cyclization/arrangement products from various ERG7H234X mutants demonstrated the catalytic plasticity of the enzyme. Specifically, subtle changes in the active site affect both the stability of the cation-π interaction and generate product diversity.