A series of aminotriarylmethane dyes were examined for binding to the nicotinic acetylcholine receptor (AChR) from Torpedo californica. Several compounds were found to bind to the noncompetitive antagonist site of the AChR as demonstrated by inhibition of [3H]phencyclidine binding; apparent KD values ranged from 50 nM to >100 microM. One dye with high affinity, crystal violet, revealed a greater than 200-fold fluorescence enhancement upon binding the AChR. Using fluorescence to measure binding, we determined that one crystal violet bound per receptor with a dissociation constant of 100 nM; in the presence of the agonist carbamylcholine this value decreased to 10 nM. The KD for [3H]acetylcholine binding likewise was decreased in the presence of crystal violet. These results are consistent with preferential binding of crystal violet to the desensitized conformation of the AChR. Crystal violet binding blocked agonist-induced 22Na ion efflux from AChR-rich vesicles. It is concluded that crystal violet and other dyes of similar structure bind to the high-affinity noncompetitive antagonist site of the AChR associated with the channel lumen. Because of their optical properties, crystal violet and several of the other homologous dyes are likely to be useful ligands for further characterization of the AChR channel. Structure-activity comparison of the various dyes suggests the importance of nonquaternary nitrogens in binding the pore. Additional steric bulk on amines or at meta positions increase or have neutral effect on affinity, suggesting that steric considerations alone do not limit high affinity for the binding site.