Picrotoxin (PTX) is a noncompetitive antagonist of many ligand-gated ion channels, with a site of action believed to be within the ion-conducting pore. In the A-type gamma-aminobutyric acid receptor, a threonine residue in the second transmembrane domain is of particular importance for the binding of, and ultimate inhibition by, PTX. To better understand the relationship between this residue and the PTX molecule, we mutated this threonine residue to serine, valine, and tyrosine to change the structural and biochemical characteristics at this location. The known subunit stoichiometry of the A-type gamma-aminobutyric acid receptor allowed us to create receptors with anywhere from zero to five mutations. With an increasing number of mutated subunits, each amino acid substitution revealed a unique pattern of changes in PTX sensitivity, ultimately encompassing sensitivity shifts over several orders of magnitude. The electrophysiological data on PTX-mediated block, and supporting modeling and docking studies, provide evidence that an interaction between the PTX molecule and three adjacent uncharged polar amino acids at this position of the pore are crucial for PTX-mediated inhibition.