AbstractActive site mapping of rat liver oxidosqualene cyclase (OSC), a 78 kDa membrane‐bound enzyme, was carried out using the mechanism‐based irreversible inhibitor, [3H]29‐methylidene‐2,3‐oxidosqualene. The amino acid sequence of the radiolabeled CNBr peptide fragment showed unexpectedly high similarity to the yeast OSC, plant OSC, and bacterial squalene cyclases. Further, radio analysis established that the two adjacent Asp residues in the highly conserved region (Asp‐Asp‐Thr‐Ala‐Glu‐Ala, or DDTAEA) were equally labeled by the irreversible inhibitor. This result provided the first information on the structural details of the active site of OSC, and showed for the first time the ancient lineage of this vertebrate enzyme to ancestral eukaryotic and prokaryotic cyclases. Interestingly, the covalently‐modified DDXX(D/E) sequence of rat liver OSC showed surprising similarity to the putative allylic diphosphate binding site sequence of other terpene cyclases and prenyl transferases. The Asp‐rich motif may act as a point charge to stabilize incipient cationic charge.