The binding of agonists, antagonists, and the acetylcholinesterase inhibitor, eserine, to the nicotinic acetylcholine receptor from Torpedo californica has been monitored by the fluorescence changes of two extrinsic probes that have been covalently attached to the receptor protein. Although both probes, 5-(iodoacetamido)salicylic acid (IAS) and 4-[N-[(2-iodoacetoxy)ethyl]-N-methylamino]-7-nitrobenz-2-oxa-1,3-diaz ol e (IANBD) react with sulfhydryl groups, they do not react at the same location. The conditions for IAS labeling and competition studies have shown that, following reduction of the receptor, this fluorophore reacts with the same cysteines on each of the two alpha subunits that may be labeled by the alkylating agonist, [3H]bromoacetylcholine. The fluorescence of this probe is sensitive to the binding of agonists and competitive antagonists to two high-affinity sites on the receptor. IANBD does not react with the same cysteines as IAS, and its fluorescence is unchanged by the high-affinity binding of agonists and antagonists. The fluorescence of this probe is, however, specifically and saturably enhanced by the binding of agonists to distinct low-affinity sites. Heterogeneity in the NBD fluorescence changes induced by the bis-quaternary agonist, suberyldicholine, has indicated that the stoichiometry of low-affinity sites is also two per receptor. Stopped-flow studies of agonist binding to receptor preparations that had been doubly labeled by both fluorophores demonstrate that the conformational changes detected by IAS occur on slow time scales of seconds to minutes whereas a much faster conformational change is revealed by changes in NBD fluorescence.(ABSTRACT TRUNCATED AT 250 WORDS)