Mutations of the proline residue at the 2' position (P2') within the second transmembrane (M2) domain of the gamma-aminobutyric acid(C) (GABA(C)) rho1 subunit are known to produce receptors with altered pharmacology. In the present study, P2' was mutated to alanine (rho1P2'A), phenylalanine (rho1P2'F), glycine (rho1P2'G) and serine (rho1P2'S). Mutant receptors were characterized using a range of agonists, partial agonists and antagonists. rho1P2'A, rho1P2'G and rho1P2'S receptors were less susceptible than wild-type receptors to agonist activation. Most notably, the partial agonists, (+/-)-trans-2-(aminomethyl)cyclopropanoic acid ((+/-)-TAMP) and imidazole-4-acetic acid (I4AA) were converted to antagonists at rho1P2'G and rho1P2'S receptors and the partial agonist CACA acted as an antagonist at rho1P2'S receptors. In contrast, rho1P2'F receptors were more prone to activation by agonists. A correlation was observed between the pharmacological properties of the mutant receptors and the hydrophobicity of each residue. Unlike the agonists or partial agonists, the affinity of competitive antagonists, (1,2,5,6-tetrahydropyridine-4-yl)methylphosphinic acid (TPMPA) and 4,5,6,7-tetrahydroisoxazole[4,5-c]pyridine-3-ol (THIP), did not change significantly between wild-type and mutant receptors. Thus, the results suggest that the agonist/competitive antagonist binding site(s) were not significantly affected by the mutations, but that receptor activation properties altered such that the more hydrophobic the residue at the 2' position, the more prone the receptor is to agonist activation.