The spectroscopic properties and specificity of binding of a fluorescent quaternary amine, ethidium, with acetylcholine receptor-enriched membranes from Torpedo californica have been examined. Competition binding with [3H]phencyclidine in the presence of carbamylcholine showed that ethidium binds with high affinity to a noncompetitive inhibitor site (KD = 3.6 X 10(-7) M). However, in the presence of alpha-toxin, ethidium's affinity is substantially lower (KD approximately 1 X 10(-3) M). Ethidium was also found to enhance [3H]acetylcholine binding with a KD characteristic of ethidium binding to a high-affinity noncompetitive inhibitor site. These findings indicate that ethidium binds to an allosteric site which is regulated by agonist binding and can convert the agonist sites from low to high affinity. Fluorescence titrations of ethidium in the presence of carbamylcholine yielded a similar KD (2.5 X 10(-7) M) and showed an ethidium stoichiometry of one site/acetylcholine receptor monomer. Ethidium was completely displaced by noncompetitive inhibitors such as phencyclidine, histrionicotoxin, and dibucaine. The enhanced fluorescence lifetime of the bound species showed that the increased fluorescence intensity reflects a 13-fold increase in quantum yield for the complex compared to ethidium in buffer. Fractional dissociation of ethidium with phencyclidine produced a double-exponential fluorescence decay rate with lifetime components characteristic of ethidium free in solution and bound to the receptor. These data argue that the alterations in ethidium fluorescence elicited by other ligands is due to a change in the fraction of specifically bound ethidium rather than a change in quantum yield of a pre-existing ethidium-acetylcholine receptor complex. The extent of polarization indicates that bound ethidium is strongly immobilized. The magnitude of the quantum yield enhancement and the shifts of excitation and emission maxima of bound ethidium suggest that its binding site is within a hydrophobic domain with limited accessibility to the aqueous phase.