The simultaneous action of pyridoxal 5′-phosphate (PLP) and L-homocysteine on specific [ 3H]muscimol binding to the γ-aminobutyric acid receptor on freeze-thawed, Triton-treated calf-brain synaptic membranes was examined kinetically by double inhibition analysis. PLP was found to be a pure inhibitor and L-homocysteine, a partial inhibitor, with respect to [ 3H]muscimol. Diagnostic analysis of the experimental data showed that the interaction constant (α) of the two inhibitors for the free receptor is between 0 and 1, confirming that the two inhibitors act synergistically. Further double inhibition analysis showed that no quaternary receptor-[ 3H]muscimol-homocysteine-PLP complex is formed although the ternary receptor-homocysteine-PLP complex is present. The localization and relationship of binding groups for both inhibitors is discussed as in their association with the ligand binding site.