Pentobarbital was continuously infused intracerebroventricularly (i.c.v.) at the rate of 300 μg/10 μl/h for 7 days, and withdrawal from pentobarbital was rendered 24 h after the stopping of the infusion. To eliminate the induction of hepatic metabolism by systemic administration of pentobarbital, an i.c.v. infusion model of tolerance to and withdrawal from pentobarbital was used. Little is known about the functional modulation of the G protein α-subunits at the molecular level. The effects of continuous infusion of pentobarbital on the modulation of G protein α-subunits mRNA were investigated by using in situ hybridization study. In situ hybridization showed that the level of Gαs mRNA was increased in the septum and brainstem, and the level of Gαo mRNA was elevated in the cortex during the pentobarbital withdrawal. The level of Gαi mRNA was significantly elevated in almost all area of brain during the pentobarbital withdrawal. These results suggest that region-specific changes of G protein α-subunit mRNA were involved in the withdrawal from pentobarbital, whereas α-subunit is not so highly involved in the pentobarbital tolerance.