The agonist binding site of the nicotinic acetylcholine receptor has a loop-based structure, and is formed by residues located remotely to each other in terms of primary structure. Amino acid residues in sites δ57 and δ59, and the equivalent residues in the ε subunit, have been identified as part of the agonist binding site and designated as loop D. The effects of point mutations in sites δ57, δ59, ε55 and ε57 on agonist binding and channel gating were studied. The mutated receptors were expressed transiently in HEK 293 cells and the currents were recorded using the cell-attached single-channel patch clamp technique. The results demonstrate that the mutations mainly affect channel gating with the major portion of the effect due to a reduction in the channel opening rate constant. For both the δ57/ε55 and the δ59/ε57 site, a mutation in the ε subunit had a greater effect on channel gating than a mutation in the δ subunit. In all instances, agonist binding was affected to a lesser degree than channel gating. Previous data have placed the δ57 and δ59 residues in or near the agonist binding pocket. The data presented here suggest that these two residues (and the homologous sites in the ε subunit) are not involved in specific interactions with the nicotinic agonist and that they affect the activation of the nicotinic receptor by shaping the overall structure of the agonist binding site.