Clinical concentrations of pentobarbital inhibit the alpha-amino-3-hydroxy-5- methyl-4-isoxazole propionic acid (AMPA)-selective glutamate receptor (GluR) channels. Recently, the AMPA-selective GluR channels that contained the alpha 2 subunit were shown to be more sensitive to pentobarbital block than those without the alpha 2 subunit. Here we demonstrated that replacement by glutamine of the arginine residue in putative transmembrane segment M2 of the alpha 2 subunit (mutation alpha 2-R586Q) drastically reduced the pentobarbital sensitivity of the alpha 2 heteromeric channel to the level comparable to those of the alpha 1 and alpha 2-R586Q homomeric channels. These results suggest that the arginine residue in segment M2 of the alpha 2 subunit is the critical determinant of the sensitivities of the AMPA-selective GluR channels to pentobarbital.