Abstract: γ-Aminobutyric acid (GABA) is the principal inhibitory neurotransmitter in the central nervous system. GABA receptors are ligand-gated chloride ion channels formed by five homologous subunits around the central channel pore. Structural aspects of the noncompetitive antagonist (NCA) site are examined here by expressing each of the 24 amino acids in the transmembrane 2 (M2) and flanking region of the pore as individual cysteine mutants plus serine, alanine or phenylalanine at some specific positions using the β3 homomeric receptor for symmetry to facilitate interpretations. The positions are given as index numbers for the M2 region. Disulfide trapping showed facile subunit cross-linking (dimer formation) on oxidation with copper phenanthroline for cysteines at -1', 2'(very weak), 6', 9', 14', and 16', but 13' and 17'-20' all formed disulfide bonds spontaneously. There was little or no dimer formation at positions 11', 12' and 15'. Reduction with dithiothreitol (DTT) cleaves the spontaneously-formed or induced dimers except those at 6' and 13'. Several major insecticides, including α-endosulfan, lindane and fipronil, and the botanical picrotoxinin act as NCAs. Binding of two NCA radioligands ([3H]EBOB and [3H]BIDN) was dramatically reduced with 8 of the 15 mutated positions in the cytoplasmic half of the pore (-4' to 10'). The six NCAs listed above plus TBPS of widely-diverse structures may fit the pore region modeled as an α-helix and block the channel on hydrogen bonding with the T6' hydroxyl and hydrophobic interactions with A2', T6' and L9' methylene and methyl substituents. The extracellular half of the receptor (11' to 20') also actively participates in its functions. [3H]EBOB binding is impeded by cysteines at 13', 14', 16', 17' and 19' but in the case of 17' and 19', this is reversed with DTT. Changes in the extracellular end of the channel gate can modulate or almost block NCA binding. A possible β-conformation at region 17'-20' is suggested by the spontaneous disulfide formation pattern. The Zn2+ site identified by an H17'A mutation is coupled with the [3H]EBOB site. The NCA site, conserved between mammals and insects, confers toxicant sensitivity and specificity while other subunits in the natural heteromeric receptor are proposed to modulate selectivity and species specificity.