Dried Chrysanthemum morifolium flowers have traditionally been used in Korea for the treatment of insomnia. This study was performed to investigate whether the ethanol extract of Chrysanthemum morifolium flowers (EFC) enhances pentobarbital-induced sleep behaviors. EFC prolonged sleep time induced by pentobarbital similar to muscimol, a GABA(A) receptors agonist. EFC also increased sleep rate and sleep time when administrated with pentobarbital at a subhypnotic dosage. Both EFC and pentobarbital increased chloride (Cl(-)) influx in primary cultured cerebellar granule cells. EFC increased glutamic acid decarboxylase (GAD) expression levels, but had no effect on the expression of α1-, β2-, and γ2-subunits of the GABA(A) receptor in the hippocampus of a mouse brain. This is in contrast to treatment with pentobarbital, which showed decreased α1-subunit expression and no change in GAD expression. In conclusion, EFC augments pentobarbital-induced sleep behaviors; these effects may result from Cl(-) channel activation.