The nonsense codon suppression method for unnatural amino acid incorporation has been applied to intact Cells and combined with electrophysiological analysis to probe structure-function relations in the nicotinic acetylcholine receptor, Functional receptors were expressed in Xenopus oocytes when tyrosine and phenylalanine derivatives were incorporated at positions 93, 190, and 198 in the binding site of the a subunit, Subtle changes in the structure of an individual side chain produced readily detectable changes in the function of this large channel protein, At each position, distinct features of side chain structure dominated the dose-response relation, probably by governing the agonist-receptor binding.