The anticonvulsant α-ethyl, α-methyl-γ-thiobutyrolactone (αEMTBL) potentiates the response to a submaximal concentration of glycine produced by receptors composed of human glycine α1-subunits but reduces the response of receptors composed of rat glycine α3-subunits. Both the potentiating and blocking actions of αEMTBL are reduced by higher concentrations of glycine. The subunit specificity of αEMTBL block is conferred by a residue in the second membrane-spanning region (M2), which is alanine in the α3-subunit (A254) and glycine in the α1-subunit. The mutation A254G in the α3-subunit removes blocking by αEMTBL and reveals potentiation. Picrotin, a picrotoxinin analog, blocks responses of receptors composed of either α1 or α3-subunits. Blocking of α3 receptors by picrotin is reduced in the presence of αEMTBL, indicating that the mechanisms interact at some point, but the mutation α3 A254G does not remove block by picrotin. However, mutation of a nearby residue α3 T258F does remove block by picrotin, picrotoxinin and αEMTBL. These observations suggest that αEMTBL and picrotin act on glycine α3 receptors to produce block by an allosteric mechanism that involves overlapping sets of residues in the M2 region. Coexpression of the α3-subunit with the β-subunit of the glycine receptor also removes block by αEMTBL and reveals potentiation, suggesting that receptors containing either α3 or α1 glycine receptor subunits are potentiated in the adult brain.