The subunits that constitute the โฅ-aminobutyric acid (GABA) C receptors of retinal neurons form a unique subclass of ligand-gated chloride channels that give rise to sustained GABA-evoked currents that exhibit slow offset (deactivation) kinetics. We exploited this property to examine the molecular mechanisms that govern the disparate response kinetics and pharmacology of perch GABA 1B and 2A subunits expressed in Xenopus oocytes. Using a combination of domain swapping and site-directed mutagenesis, we identified the residues at amino acid position 320 in the second transmembrane domain as an important determinant of the receptor kinetics of GABA C receptors. When the site contains a proline residue, as in wild-type 1 subunits, the receptor deactivates slowly; when serine occupies the site, as in wild-type 2 subunits, the time course of deactivation is more rapid. In addition, we found that the same site also altered the pharmacology of GABA receptors, e.g., when the serine residue of the 2A receptor was changed to proline, the response of the mutant receptor to imidazole-4-acetic acid (I4AA) mimicked that of the 1B receptor. However, despite gross changes in receptor pharmacology, the apparent binding affinity for the drug was not significantly altered. These findings provide further evidence that the second transmembrane domain is involved in the gating mechanism that governs the response properties of the various receptor subunits. It is noteworthy that the proline residue in native 1 subunits and the serine residue of 2 subunits are well conserved in all species, a good indication that the presence of multiple GABA subunits serves to generate GABA C receptors that display the wide range of response kinetics observed on various types of retinal neurons.