These studies demonstrated that 5-ethyl-5-(3-hydroxyl-1-methyl-butyl)-barbituric acid (PB-OH), a major metabolite of pentobarbital, antagonized pentobarbital-induced narcosis in both naive and pentobarbital tolerant mice. In PB-OH pretreated mice, the sleeping time induced by sodium pentobarbital was significantly shorter than that of the saline control animals. However, PB-OH failed to modify the pentobarbital-induced hypothermia. The findings also demonstrated that hepatic microsomal enzyme activity and half lives of pentobarbital and PB-OH in both plasma and brain were not modified by the pretreatment of PB-OH. The specific antagonistic effect of PB-OH appears to be a direct effect on sites in the CNS.