The nicotinic acetylcholine receptor exhibits at least four different affinity states for agonists such as acetylcholine. In order to identify the structural changes occurring at or near the agonist binding site during the allosteric transitions, three photoactivatable compounds designed to display agonist activity were synthesized. Inhibition constants of these compounds for the cholinergic and the noncompetitive blocker binding sites were determined for the resting and the desensitized states of the receptor. Among these probes, two ligands, AC5 and AC7, displayed a high affinity for the agonist binding site and were poorly recognized by the binding site for noncompetitive blockers. Electrophysiological experiments revealed that these ligands behaved as agonists at low concentrations. We used these two compounds in photolabeling experiments and observed that they were able to inactivate the agonist binding site. Up to 50% of these sites were irreversibly inhibited, depending on the ligand, the irradiation conditions, and the selected receptor state. The compound with the most interesting properties (high affinity and selectivity for the acetylcholine binding site, as well as agonist activity and high photolabeling yield) is AC5, a structural analogue of the fluorescent agonist dansyl-C6-choline, which has been previously used to characterize the different states of the nicotinic receptor. After radioactive synthesis, [3H]AC5 was shown to label all four receptor subunits, in a protectable manner. This radioligand, thus, appears suitable for investigation of the dynamics of allosteric transitions occurring at the activated acetylcholine binding site.