Poly(A)+ RNA from bovine retina expressed gamma-aminobutyric acid (GABA)-activated membrane current responses in Xenopus oocytes, consisting of two pharmacologically distinct components. One component (IG-Aret) was mediated by GABAA receptors, and the other component (KG-BR) by atypical GABA receptors that were resistant to inhibition by bicuculline and insensitive to activation by baclofen. To further characterize the bicuculline/baclofen-insensitive GABA receptors, electrical recordings were made measuring the sensitivity of IG-BR to two Cl- channel inhibitors, t-butylbicyclophosphorothionate (TBPS) and picrotoxin. For purposes of comparison, effects of TBPS and picrotoxin were also assayed on currents mediated by GABAA receptors expressed in oocytes by rat cerebral cortex RNA (IG-Actx). The main finding of this study was that TBPS was a surprisingly weak inhibitor of IG-BR, whereas IG-Actx was potently suppressed. Assays on maximum responses indicated that IG-Actx was at least 500 times more sensitive to TBPS than was IG-BR (IC50 values of approximately 0.2 microM and greater than 50 microM, respectively). Moreover, inhibition of IG-Actx by micromolar concentrations of TBPS was largely insurmountable, whereas the weak inhibitory effects on IG-BR showed strong dependence on agonist concentration. For example, 10 microM TBPS reduced maximum IG-Actx by greater than 90%, an effect that was not significantly reversed by 10-fold increases in the concentration of agonist. In contrast, the same concentration of TBPS caused a 2-fold increase in the EC50 for IG-BR but had only marginal (less than 5%) inhibitory effects on maximum responses. Picrotoxin inhibited both types of current, but assays on maximum responses indicated that IG-Actx was approximately 30 times more sensitive than IG-BR (IC50 values of approximately 1 and 30 microM, respectively). Inhibitory effects of picrotoxin on IG-BR again showed strong dependence on agonist concentration, but in this case there was also a clear insurmountable component. Comparisons between IG-Actx and IG-Aret suggested that GABAA receptors expressed by either brain or retina RNA showed approximately the same sensitivity to TBPS and picrotoxin. Our experiments indicate that the bicuculline/baclofen-insensitive GABA receptors expressed by retina RNA differ markedly from GABAA receptors in their sensitivity to TBPS and picrotoxin. Defining the structural features responsible for these differences at the molecular level will provide a further means of investigating the complex mechanisms underlying interactions between inhibitors and GABA-activated Cl- channels.