The nonsense codon suppression technique was used to incorporate o-nitrobenzyl cysteine or o-nitrobenzyl tyrosine (caged Cys or Tyr) into the 9' position of the M2 transmembrane segment of the -subunit of the muscle nicotinic ACh receptor expressed in Xenopus oocytes. The caged amino acids replaced an endogenous Leu residue that has been implicated in channel gating. ACh-induced current increased substantially after ultraviolet (UV) irradiation to remove the caging group. This represents the first successful incorporation of caged Cys into a protein in vivo and the first incorporation of caged amino acids within a transmembrane segment of a membrane protein. The bulky nitrobenzyl group does not prevent the synthesis, assembly, or trafficking of the ACh receptor. When side chains were decaged using 1-ms UV light flashes, the channels with caged Cys or caged Tyr responded with strikingly different kinetics. The increase in current upon photolysis of caged Cys was too rapid for resolution by the voltage-clamp circuit [time constant () <10 ms], whereas the increase in current upon photolysis of caged Tyr was dominated by a phase with  ~500 ms. Apparently, the presence of a bulky o-nitrobenzyl Tyr residue distorts the receptor into an abnormal conformation. Upon release of the caging group, the receptor relaxes, with  ~500 ms, into a conformation that allows the channel to open. Tyr at the 9' position of the -subunit greatly increases the ability of ACh to block the channel by binding within the channel pore. This is manifested in two ways. 1) A "rebound," or increase in current, occurs upon removal of ACh from the bathing medium; and 2) at ACh concentrations >400 µM, inward currents are decreased through the mutated channel. The ability to incorporate caged amino acids into proteins should have widespread utility.