Abstract: Tolerance to and withdrawal from pentobarbital were induced in rats by continuous intracerebroventricular infusion via subcutaneously implanted osmotic minipumps. In situ hybridization of GABAA receptor α1- and β3-subunit mRNA was conducted using synthetic 3′- end 35S-dATP-labeled oligodeoxynucleotide probes. Results were quantified by film densitometry. In animals that were tolerant to pentobarbital, levels of α1-subunit mRNA were decreased in hippocampus, superior colliculus, and inferior colliculus, but levels of β3-subunit mRNA were not affected. Dramatically increased levels of GABAA receptor subunit mRNA were observed in animals 24 h after withdrawal from chronic pentobarbital treatment. These increases occurred in cerebral cortex and cerebellum for the α1 subunit and in cerebral cortex only for the β3-subunit. These data provide further support to the structural and pharmacological GABAA receptor heterogeneity in discrete brain areas. The observed changes of subunit expression may underlie, at least in part, the receptor up- and down-regulation observed in receptor ligand binding studies.