Lanosterol synthase [(S)-2,3-epoxysqualene mutase (cyclizing, lanosterol forming), EC 5.4.99.7], the enzyme from Saccharomyces cerevisiae which catalyzes the complex cyclization/rearrangement step in sterol biosynthesis, was overexpressed in baculovirus-infected cells and purified to homogeneity in three steps. Using pure enzyme the kinetics of cyclization were determined using Michaelis-Menten analysis for 2,3-oxidosqualene (1) and two analogs in which the C-6 methyl was replaced by H (3) or Cl (4). The measured V-max/K-M ratios for 1, 3, and 4 were found to be 138, 9.4, and 21.9, respectively, a clear indication that oxirane cleavage and cyclization to form the A-ring are concerted, since the nucleophilicity of the proximate double bond influences the rate of oxirane cleavage. No catalytic metal ions could be detected in purified lanosterol synthase by atomic absorption analysis. Site-directed mutagenesis studies of each of the six strongly conserved aspartic acid residues (D --> N mutation) and each of the nine conserved glutamic acid residues (E --> Q) revealed that only one, D456, is essential for catalytic function of the enzyme. The essential D456 residue is a likely candidate for electrophilic (specifically protic) activation of the oxirane function.