The nicotinic acetylcholine receptor possesses an agonist binding site on each of the two alpha-subunits and an allosterically coupled noncompetitive inhibitor (NCI) site. The spatial relationships between these sites have been determined by fluorescence energy transfer employing lifetime and steady-state techniques with two donor-acceptor pairs. 6-(5-Dimethylaminonaphthalene-1-sulfonamido)hexanoic acid-beta-(N-trimethylammonium)ethyl ester (dansyl-C6-choline, an agonist) and bis(choline)-N-(4-nitrobenzo-2-oxa-1,3-diazol-7-yl)-iminodiprop rionate (BCNI, a competitive antagonist) were employed as energy donors bound to the agonist sites. Ethidium was employed as a specific probe of the NCI site and served as the energy acceptor for both donors. Under steady-state conditions, energy transfer was measured by monitoring BCNI fluorescence as a function of occupancy of ethidium. Changes in acceptor occupancy were achieved by titrating acetylcholine receptor-donor-acceptor complexes with phencyclidine, a nonfluorescent NCI ligand. Extrapolation of the data to 100% acceptor occupancy yielded a transfer efficiency of 38% for the BCNI-ethidium pair. In the second method, the transfer efficiency of the dansyl-C6-choline-ethidium pair was determined by analysis of the reduction of the donor-excited state fluorescence lifetime. The nanosecond decay rates for dansyl-C6-choline measured in the presence of phencyclidine are characterized by two lifetimes (tau 1 = 6.7; tau 2 = 17.1 ns) with an amplitude ratio, alpha 1/alpha 2 = 2.3. In the presence of ethidium, the two lifetimes were proportionally diminished while retaining a comparable ratio of amplitudes. Displacement of ethidium from the NCI site by phencyclidine restored the two lifetimes to their original values. These data indicate that the donors bound to the two agonist sites transferred energy with similar efficiencies to the acceptor. Thus, the lifetime data suggest that the NCI site is approximately equidistant from each of the agonist sites. The corrected efficiency of donor quenching by this method was 34%, a value in close accord with the steady-state measurements. The distance between the agonist sites and the NCI site was calculated to be between 21-35 A for the BCNI/ethidium pair and 22-40 A for the dansyl-C6-choline/ethidium pair. Consideration of these distances with respect to the molecular dimensions of the receptor and location of the agonist sites suggests a location for the NCI site near the ion channel at the extracellular surface of the membrane bilayer.