We investigated whether the interoceptive discriminative stimulus (IDS) arising from ethanol withdrawal was related to decreased activity of the gamma-aminobutyric acid (GABA) system by determining whether the sensitivity of rats to the GABA antagonists was altered by chronic treatment with ethanol. Rats were trained to obtain food reward by responding on one lever following pentylenetetrazol (PTZ) and the other lever following saline. Whereas all of the trained rats selected the PTZ-appropriate lever after PTZ, no more than 50% of them selected this lever following an optimum dose of bicuculline or picrotoxinin. After either saline or diazepam (5 mg/kg), all of the rats selected the saline-appropriate lever. Ethanol (0.24 mol/kg/day) was then administered to the rats for 4 days via a nutritionally balanced liquid diet. Between 48 and 96 hours postethanol, 30% of the rats selected the PTZ-appropriate lever following saline, whereas selection of this lever was increased to 80% following either bicuculline or picrotoxinin. Thus, further antagonism of GABAergic activity increased the subjective effect of ethanol withdrawal. These data support the hypothesis that the PTZ-like IDS produced during withdrawal from ethanol is related to an ethanol-induced deficit in the activity of the GABA-benzodiazepine receptor-coupled chloride channel.