The γ-aminobutyric acid-A (GABA(A)) receptor complex is allosterically modulated by a variety of substances, some of clinical importance. Barbiturates and neurosteroids augment GABA-currents and also directly gate the channel. A variety of γ-butyrolactone analogues also modulate GABA- induced currents, with some potentiating and others inhibiting. Because several γ-thiobutyrolactone analogues have biphasic effects on GABA currents, experiments with wild-type and picrotoxinin-insensitive GABA(A) receptors were performed to analyze whether some γ-thiobutyrolactones interact with two distinguishable sites on the GABA(A) receptor. β-Ethyl- β-methyl-γ-thiobutyrolactone inhibited GABA-induced currents at low concentrations (0.001-1 mM), but potentiated GABA-induced currents at higher concentrations (3-10 mM) in wild-type α1β2γ2-subunit containing ionophores. The related α-ethyl-α-methyl-γ-thiobutyrolactone potentiated submaximal GABA currents in wild-type receptors at both low and high concentrations (0.1-10 mM). Mutations in the second transmembrane domain of α1, β2, or γ2 conferred picrotoxinin-insensitivity onto GABA(A) receptor complexes. When these mutated α1, β2, or γ2 subunits were incorporated into the receptor complex, β-ethyl-β-methyl-γ-thiobutyrolactone potentiated GABA currents over the entire concentration range (0.1-10 mM). Neither the potentiating activity nor the EC50 of α-ethyl-α-methyl-γ- thiobutyrolactone changed in the mutant receptors. Further studies demonstrated that the mutations did not affect the EC50 of chlordiazepoxide or phenobarbital. These and our earlier results identify a modulatory site on the GABA(A) receptor distinct from that interacting with barbiturates, benzodiazepines, and steroids. Additionally, they show that the γ- butyrolactones probably interact at two different sites on the ionophore to produce opposite effects on GABA-mediated current.