The metabolic routes of valproic acid (VPA) were studied by i.p. administration of the mono-unsaturated and hydroxylated metabolites to rats. Conjugation with glucuronic acid was a major metabolic route for VPA and its metabolites. Conjugation with glycine was a minor route for VPA, but was of more importance with the unsaturated metabolites. The hydroxylated metabolites, which were further oxidized to oxo-derivatives and subsequently to the dicarboxylic acids, were not metabolically dehydrated to form unsaturated metabolites. Multiple metabolic pathways, including dehydrogenation, isomerization, hydration, hydroxylation, reduction and epoxidation were inferred from the metabolites obtained after dosage of the unsaturated metabolites. Six dien-VPA metabolites were detected in VPA-treated rats, four of which are present in patients. It was concluded that 3-en-VPA and 4-en-VPA pathways, originating through dehydrogenation, are distinct from the omega- and omega-1-hydroxylation pathways. Enzyme induction from co-administration of phenobarbital caused enhancement of the minor omega-1-oxidation pathway, yet the largest effect on clearance came from increases in glucuronidation. Mitochondrial processes were unaffected, resulting in decreased contribution to the total clearance.