Long-term social isolation stress has been shown to cause a decrease in pentobarbital (PB)-induced sleeping time in mice. In the present study, to clarify whether the GABA A/benzodiazepine (BZD) receptor system is involved in the decrease in the hypnotic activity of PB by social isolation stress, we examined the effects of BZD receptor ligands on the PB-induced sleep in group-housed and socially isolated mice. Moreover, we also tested whether social isolation stress affects the ability of GABA to stimulate 36Cl − uptake or the modulatory effect of diazepam and PB on GABA-induced stimulation of 36Cl − uptake into synaptoneurosomes prepared from mouse brain. Social isolation stress significantly decreased the PB-induced sleeping time in mice. The BZD receptor agonist diazepam (0.1–0.8 mg/kg, i.p.) dose-dependently prolonged PB sleep in group-housed and isolated mice, but the effect was weaker in isolated mice. In contrast, FG7142 (5–10 mg/kg, i.p.), a BZD receptor inverse agonist, shortened the sleep in group-housed but not in isolated mice. Flumazenil (16.5–33 nmol, i.c.v.), a selective BZD receptor antagonist, caused PB sleep in isolated mice to return to the level of group-housed mice, at the dose that antagonized the effects of diazepam and FG7142 on PB sleep in group-housed mice. However, this antagonist alone produced no effect on PB sleep in group-housed mice. Social isolation stress decreased the ability of GABA (0.6–200 μM) to stimulate 36Cl − uptake into synaptoneurosomes but this stress had no effect on PB- and diazepam-induced enhancement of GABA-stimulated 36Cl − uptake. These results suggest that endogenous substance(s) with an inverse BZD receptor agonist-like property and the changes in the ability of GABA to stimulate chloride ion channels are involved in the decrease in the hypnotic activity of pentobarbital following social isolation stress.