Desformylflustrabromine (dFBr) is a positive allosteric modulator (PAM) of α4β2 and α2β2 nAChRs that, at concentrations >1 µM, also inhibits these receptors and α7 nAChRs. However, its interactions with muscle-type nAChRs have not been characterized, and the locations of its binding site(s) in any nAChR are not known. We report here that dFBr inhibits human muscle (αβεδ) and Torpedo(αβγδ) nAChR expressed in Xenopusoocytes with IC50values of ∼1 μM. dFBr also inhibited the equilibrium binding of ion channel blockers to TorpedonAChRs with higher affinity in the nAChR desensitized state ([3H]phencyclidine; IC50= 4 μM) than in the resting state ([3H]tetracaine; IC50= 60 μM), whereas it bound with only very low affinity to the ACh binding sites ([3H]ACh, IC50= 1 mM). Upon irradiation at 312 nm, [3H]dFBr photoincorporated into amino acids within the TorpedonAChR ion channel with the efficiency of photoincorporation enhanced in the presence of agonist and the agonist-enhanced photolabeling inhibitable by phencyclidine. In the presence of agonist, [3H]dFBr also photolabeled amino acids in the nAChR extracellular domain within binding pockets identified previously for the nonselective nAChR PAMs galantamine and physostigmine at the canonical α-γinterface containing the transmitter binding sites and at the noncanonical δ-βsubunit interface. These results establish that dFBr inhibits muscle-type nAChR by binding in the ion channel and that [3H]dFBr is a photoaffinity probe with broad amino acid side chain reactivity.