Many pharmacological and physiological effects of ethanol have been postulated to be mediated via GABA-containing neuronal systems in the CNS. The GABA receptor exists as part of a complex containing receptors for benzodiazepines (BDZ), barbiturates and the chloride ion channel. To examine the effects of ethanol on this complex, we have studied binding properties of 35-S-t-butylbicyclophosphorothionate (35-S-TBPS), a ligand which interacts with the regulatory site of the chloride ion channel, in membranes of cortex and cerebellum of C57B1 mice in the presence and absence of ethanol. In the presence of bromide ion in vitro, ethanol produced a dose-dependent inhibition of TBPS binding, but did not potentiate inhibition of TBPS binding by GABA or pentobarbital. In the presence of chloride ion ethanol was less potent at inhibiting TBPS binding. However, under these conditions, GABA and pentobarbital had biphasic effects on TBPS binding in cortex, and low concentrations of ethanol reduced the stimulatory effects of these compounds on TBPS binding.