The binding of d-tubocurarine and several of its analogs to the mouse nicotinic acetylcholine receptor (AChR) was measured by competition against the initial rate 125I-alpha-bungarotoxin binding to BC3H-1 cells. The changes in affinity due to methylation or halogenation at various functional groups on d-tubocurarine was measured to both the high affinity (alphagamma-site) and the low affinity site (alphadelta-site). We show that quaternization by methylation of the 2'-N ammonium group enhances the affinity for both the acetylcholine binding sites of mouse AChR, whereas this change does not affect affinity for the Torpedo AChR sites. The effect of N-methylation suggests the presence of interactions with the ammonium moiety that cannot be readily attributed to the known conserved residues thought to stabilize this functional group. Methylation of both the 7'- and 12'-phenols produced net affinity changes at both sites. The changes resulted from contributions at both the 7'- and the 12'-positions; however, these effects were dependent on whether the ammoniums were also methylated. Substitution of bromine or iodine at the 13'-position decreased the affinity considerably to the high affinity alphagamma-site of mouse AChR, whereas the affinity for the Torpedo alphagamma-site was slightly increased. Furthermore, binding to the mouse AChR was unaffected by the conformational state, whereas these ligands strongly preferred the desensitized conformation of the Torpedo AChR. Comparison of binding changes upon 13'-halogenation to the changes in amino acid residues at the ACh binding sites of the mouse and Torpedo AChR shows mouse residue Ile-gamma116 as likely to be involved in interacting with the 13'-position of d-tubocurarine. It is predicted that this residue is involved in the conformational equilibrium between the resting and desensitized conformations.