Although ␥-aminobutyric acid type A receptor agonists and antagonists bind to a common site, they produce different conformational changes within the site because agonists cause channel opening and antagonists do not. We used the substituted cysteine accessibility method and two-electrode voltage clamping to identify residues within the binding pocket that are important for mediating these different actions. Each residue from ␣ 1 T60 to ␣ 1 K70 was mutated to cysteine and expressed with wild-type ␤ 2 subunits in Xenopus oocytes. Methanethiosulfonate reagents reacted with ␣ 1 T60C, ␣ 1 D62C, ␣ 1 F64C, ␣ 1 R66C, ␣ 1 S68C, and ␣ 1 K70C. ␥-Aminobutyric acid (GABA) slowed methanethiosulfonate modification of ␣ 1 F64C, ␣ 1 R66C, and ␣ 1 S68C, whereas SR-95531 slowed modification of ␣ 1 D62C, ␣ 1 F64C, and ␣ 1 R66C, demonstrating that different residues are important for mediating GABA and SR-95531 actions. In addition, methanethiosulfonate reaction rates were fastest for ␣ 1 F64C and ␣ 1 R66C, indicating that these residues are located in an open, aqueous environment lining the core of the binding pocket. Positively charged methanethiosulfonate reagents derivatized ␣ 1 F64C and ␣ 1 R66C significantly faster than a negatively charged reagent, suggesting that a negative subsite important for interacting with the ammonium group of GABA exists within the binding pocket. Pentobarbital activation of the receptor increased the rate of methanethiosulfonate modification of ␣ 1 D62C and ␣ 1 S68C, demonstrating that parts of the binding site undergo structural rearrangements during channel gating.