Previous structural and mutagenesis studies indicate that the invariant alpha-amino and alpha-carboxyl groups of glutamate receptor agonists are engaged in polar interactions with oppositely charged, conserved arginine and glutamate residues in the ligand-binding domain of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor. To examine the role of these residues (R507 and E727 in the GluR-D subunit) in the discrimination between agonists and antagonists, we analyzed the ligand-binding properties of homomeric GluR-D and its soluble ligand-binding domain with mutations at these positions. Filter-binding assays using [3H]AMPA, an agonist, and [3H]Ro 48-8587, a high-affinity antagonist, as radioligands revealed that even a conservative mutation at R507 (R507K) resulted in the complete loss of both agonist and antagonist binding. In contrast, a negative charge at position 727 was necessary for agonist binding, whereas the isosteric mutation, E727Q, abolished all agonist binding but retained high-affinity binding for [3H]Ro 48-8587, displaceable by 7,8-dinitroquinoxaline-2,3-dione. Competition binding studies with antagonists representing different structural classes in combination with ligand docking experiments suggest that the role of E727 is antagonist-specific, ranging from no interaction to weak electrostatic interactions involving indirect and direct hydrogen bonding with the antagonist molecule. These results underline the importance of ion pair interaction with E727 for agonist activity and suggest that an interaction with R507, but not with E727, is essential for antagonist binding.