The effect of phenobarbital on the potential hepatotoxicity of E-2-en-valproate (E-2-en-VPA; trans-2-en-VPA) and VPA was studied in young male Sprague-Dawley rats. E-2-en-VPA and VPA were administered daily at 750 mg/kg i.p. (divided into three doses a day) for 7 consecutive days. Phenobarbital was coadministered i.p. once daily at 100 mg/kg for 2 days, followed by daily injections of 50 mg/kg for the subsequent days of the treatment period. Additional groups of rats were treated with phenobarbital alone or received once daily administration of 4-en-VPA (100 mg/kg), a potentially hepatotoxic metabolite of VPA. Clinical chemistry data were studied before and after the period of treatment. Furthermore, drug and metabolite levels were analyzed by gas chromatography-mass spectrometry. Treatment with VPA and phenobarbital resulted in deaths and histopathological liver alterations, such as marked microvesicular steatosis and degenerative lesions, whereas no death and hepatotoxicity occurred in rats treated with E-2-en-VPA and phenobarbital. Furthermore, hyperammonemia was recorded in VPA- but not E-2-en-VPA-treated rats. In comparison to treatment with VPA or E-2-en-VPA alone, combined treatment with phenobarbital markedly reduced plasma levels of the parent drugs and metabolites originating from beta-oxidation, but, in case of VPA, increased metabolites originating from omega-oxidation. Plasma levels of 4-en-VPA were increased by phenobarbital in VPA-treated rats, but 4-en-VPA was not detectable in rats treated with E-2-en-VPA. The most severe alterations in functional and morphological liver parameters were found in rats treated with 4-en-VPA. In these animals, the extent of steatosis was significantly correlated with plasma levels of 4-en-VPA, but not its major metabolite 2,4-dien-VPA. Plasma levels of 4-en-VPA or its major metabolite 2,4-dien-VPA in rats without steatosis were markedly higher than levels of these compounds in VPA-treated rats with steatosis, suggesting that 4-en-VPA and 2,4-dien-VPA are not critically involved in the hepatotoxic effects of VPA. The data substantiate that E-2-en-VPA is less hepatotoxic than VPA and may thus offer advantages for antiepileptic therapy.