Huperzine A, an alkaloid isolated from Huperzia serrata was found to reversibly inhibit acetylcholinesterases (EC 3.1.1.7) and butyrylcholinesterases (EC 3.1.1.8) with on- and off-rates that depend on both the type and the source of enzyme. Long-term incubation of high concentrations of purified cholinesterases (1-8 microM) with huperzine A did not show any chemical modification of huperzine A. A low dissociation constant KI was obtained for mammalian acetylcholinesterase-huperzine (20-40 nM) compared to mammalian butyrylcholinesterase-huperzine (20-40 microM). This indicates that the thermodynamic stability of huperzine-cholinesterase complex may depend on the number and type of aromatic amino acid residues in the catalytic pocket region of the cholinesterase molecule.