Fluorescence spectroscopy was used to determine whether quinacrine and ethidium, two high-affinity noncompetitive inhibitors of the Torpedo acetylcholine receptor (AcChR), bind to the same loci. The ability of three nitroxide spin-labels, 5-doxylstearate (5-SAL), spin-labeled androstane (ASL), and TEMPO, to quench receptor-bound quinacrine and ethidium fluorescence was measured. When bound to a phencyclidine-displaceable site on the AcChR, quinacrine was 16.9 and 19 times more efficiently quenched than ethidium by the highly lipophilic 5-SAL and ASL, respectively. TEMPO, which has a limited ability to partition into Torpedo plasma membranes (<1%), was only twice as efficient at quenching receptor-bound quinacrine than ethidium fluorescence. The relative sensitivity of quinacrine and ethidium fluorescence to paramagnetic quenching was examined in three solvents, 1-butanol, sodium phosphate buffer, and acetonitrile, with TEMPO as a quencher. The results from the different solvents demonstrate that quinacrine fluorescence is intrinsically 1.4–3.6 times more sensitive than ethidium fluorescence to quenching by nitroxide spin-labels. Examination of the effect of high concentrations of 5-SAL on ethidium and quinacrine dissociation constants showed that quinacrine but not ethidium binding was competitively inhibited. Together, these results indicate that although quinacrine and ethidium bind in a mutually exclusive manner, the two inhibitors interact at different loci on the AcChR. Whereas the ethidium binding site is at a distance from membrane lipids, probably in or near the lumen, the quinacrine binding site appears to be at a lipid–protein interface in the transmembrane domain and at a distance from the lumen.