We have synthesized 2 alpha,3 alpha-cyclopropano-5 alpha-cholestan-3 beta-ol and tested it as a substrate and inhibitor of cholesterol oxidase. The cyclopropylsterol irreversibly inhibits cholesterol oxidase with a k(inact) = 0.010 min(-1) and K-i = 36 mu M. Efficient inactivation requires the general base His(447). TWO FAD-steroid adducts were isolated by reversed-phase HPLC. The UV/vis, fluorescence and mass spectra of the adducts suggest that the FAD cofactor acts as an electrophile in a cyclopropoxide ring-opening reaction to form a C-6-alkylated flavin (68%) and either an N-5 flavin adduct or a cyclic N-5-C-4a flavin adduct (32%). Cyclopropoxide ring-opening to form a C-6-FAD adduct represents a new approach to flavoenzyme inhibition.