The effectiveness of fluorescence quenching of pyrene-1-sulfonyl azide, a hydrophobic probe used to photo-label acetylcholine receptor (AcChR)-rich electroplax membranes [Sator, V., Gonzalez-Ros, J. M., Calvo-Fernandez, P., & Martinez-Carrion, M. (1979) Biochemistry 18, 1200], is used to study the accessibility of the covalently attached fluorophore to extramembranous quenchers as a function of occupancy of cholinergic receptor binding sites. In these membranes, binding of water-soluble cholinergic ligands to specific sites at the extracellular side affects the fluorophore located in a distant topographical area of the AcChR molecule. When a neurotransmitter analogue (carbamylcholine) is present, the susceptibility of the covalently attached fluorophore to quenching with externally added nitromethane decreases in comparison with that of the same membranes in the absence of carbamylcholine. This neurotransmitter agonist effect is, however, reversible as removal of carbamylcholine by dialysis restores the quenching effectiveness to that of resting nonliganded membranes. The presence of bound alpha-bungarotoxin produces an opposite effect to that of carbamylcholine and induces an increase in susceptibility to quenching agent. These results are interpreted in terms of long-range effects induced by occupancy of cholinergic sites which are detected by covalently bound fluorophore located at regions of the AcChR protein accessible through the lipid matrix of the Torpedo membrane. Such effects are presumably due to molecular rearrangements within the membrane-bound AcChR structure.