Arabidopsis thaliana LUP1 (At1g78970) catalyzes the cyclization of oxidosqualene into lupeol and 3β,20-dihydroxylupane (lupanediol). The stereochemical course of water addition to the lupanyl cation was studied. The X-ray crystal structure of lupanylepoxide 3,5-dinitrobenzoate established the configuration of epoxide as 20S. LiAID4 reduction of the epoxide enabled the chemical shift assignment of prochiral methyl groups at C20 of lupanediol. Correlation of these methyl groups with biosynthetic lupanediol from [1,2-13C2] acetate established the stereochemical course of water addition.