An investigation was carried out on the effect of β-cyclodextrin (β-CD), which is an excellent complexing agent, on the hypnotic potency of four kinds of barbituric acid derivatives (BAD) after intravenous and intraperitoneal administration in mice. Among BAD, hexobarbital (HBA), pentobarbital (PBA), phenobarbital (PhBA) and thiopental (TPA) were chosen. To evaluate the hypnotic potency, the sleeping lag (the time until the onset of the righting reflex from administration) and the sleeping time (the time until recovery from the loss of righting reflex) were determined. In the cases of intravenous administration at the dose of 215.3μmol/kg weight, the sleeping times were significantly shortened, but the sleeping lags were not affected by the simultaneous administration of β-CD equivalent (in molar amount) to BAD. However, the brain concentration of BAD at the time of awakening was little affected by the simultaneous administration of β-CD. The solubility of HBA in pH 7.4 phosphate buffer solution with rat serum increased sigmoidally with increasing amount of β-CD. The present results suggest that the shortening in sleeping time may be due to a decrease in the distribution of BAD to the brain, at least partly as a result of complex formation of BAD with β-CD, resulting in a shortening of the BAD-induced sleeping time.