In order to study the chronic effects of pentobarbital, a positive GABAA receptor modulator, on the inverse agonist binding of the benzodiazepine site, binding of [3H]Ro15-4513 and levels of GABAA receptor alpha 6-subunit mRNA were investigated in the brains of pentobarbital-tolerant/dependent animals, using receptor autoradiography and in situ hybridization histochemistry in consecutive brain sections. Pentobarbital was administered to rats either 60 mg/kg, i.p., once, for acute treatment, or 300 micrograms/10 microliters/h i.c.v. continuously for 6 days via osmotic minipumps to render rats tolerant to pentobarbital. Rats assigned to the dependent group were sacrificed 24 h after discontinuance of pentobarbital infusion, while those assigned to the tolerant group were sacrificed at the end of infusion. The alpha 6 subunit mRNA was increased in the tolerant group only. Diazepam-insensitive [3H]Ro15-4513 binding was increased in the cerebellar granule layer of pentobarbital-tolerant and -dependent rats. No alterations in these parameters were observed in acutely treated animals. These data suggest that chronic pentobarbital treatment induced expression of alpha 6-subunit mRNA. This was in contrast to alpha 1- and gamma 2-subunit mRNA, which in tolerant animals are unchanged, but for which withdrawal triggers a surge in levels. Because the alpha 6-subunit is a major component of the diazepam-insensitive [3H]Ro15-4513 binding site, the increased diazepam-insensitive [3H]Ro15-4513 binding implied de novo synthesis of the receptor subunit protein.