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Ligands acting on receptors are considered to induce a conformational change within the ligand-binding site by interacting with specific amino acids. In this study, tyrosine 102 (Y102) located in the GABA binding site of the ρ(1) subunit of the GABA(C) receptor was mutated to alanine (ρ(1Y102A)), serine (ρ(1Y102S)), and cysteine (ρ(1Y102C)) to assess the role of this amino acid in the action of 12 known and 2 novel antagonists. Of the mutated receptors, ρ(1Y102S) was constitutively active, providing an opportunity to assess the activity of antagonists on ρ(1) receptors with a proportion of receptors existing in the open conformational state compared to those existing predominantly in the closed conformational state. It was found that the majority of antagonists studied were able to inhibit the constitutive activity displayed by ρ(1Y102S), thus displaying inverse agonist activity. The exception was (±)-4-aminocyclopent-1-enecarboxamide ((±)-4-ACPAM) (8) not exhibiting any inverse agonist activity, but acting explicitly on the closed conformational state of ρ(1) receptors (ρ(1) wild-type, ρ(1Y102C) and ρ(1Y102A)). It was also found that the GABA antagonists were more potent at the closed compared to the open conformational states of ρ(1) receptors, suggesting that they may act by stabilizing closed conformational state and thus reducing activation by agonists. Furthermore, of the antagonists tested, Y102 was found to have the greatest influence on the antagonist activity of gabazine (SR-95531 (13)) and its analogue (SR-95813 (14)). This study contributes to our understanding of the mechanism of inverse agonism. This is important, as such agents are emerging as potential therapeutics.