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 alpha 1- and beta 3-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 alpha 1-subunit mRNA were decreased in hippocampus, superior colliculus, and inferior colliculus, but levels of beta 3-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 alpha 1 subunit and in cerebral cortex only for the beta 3-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.