The dissociation constants (Kdiss) of mono-and bis-N-substituted benzoquinolinium salts from butyrylcholinesterase were determined by the disappearance of the fluorescence of the free ligand. The Kdiss value determined by this method agreed closely with the dissociation constants (KI,s) of the free enzyme obtained by steady-state kinetic studies. The results indicate that the N-substituted benzoquinolinium salts with a strong binding ability to butyrylcholinesterase also are potent inhibitors of the esterolytic properties of the enzyme. Enzyme binding is favored by the coplanar structure and does not require a high concentration of charge on the onium function. The shape of the hydrophobic structures markedly influences binding.