Structural determinants of L-[H-3]nicotine binding to synthetic peptides comprising residues 188-207 of nicotinic acetylcholine receptor alpha subunits were invesitigated by equilibrium binding analysis. Two binding components were detected, one of low affinity (K-d similar to 1.5 mu M) that did not differ significantly among peptides and another of high affinity. The high affinity binding component was higher for the neuronal peptides (K-d = 14-23 nM) than the muscle alpha 1 peptides (K-d = 52 nM), The following nonconservative substitutions in the alpha 4 peptide resulted in a significant decrease in nicotine affinity for the peptide: Y190A, Y190D, C192G, E195A, E195-, P199A, P199-, and Y203A Substitution of alpha 4P199 with a leucine which is present in the alpha 1 sequence decreased the affinity of the alpha 4 peptide for nicotine and substitution of alpha 1L199 with a proline (alpha 4) or a glutamine (alpha 3) increased the affinity of the alpha 1 peptide. It is concluded that aromatic residues contribute to the binding site for nicotine on the alpha 4 subunit and that the residue present at position 199 partly determines differences in nicotine affinity for different alpha subunits.