Valproic acid (VPA) is a simple branched medium-chain fatty acid with expanding therapeutic applications beyond its prime anticonvulsant properties. Aims: (1) To resolve the underlying basis for the interference of valproate with the isoleucine degradative pathway and (2) to shed new light on the enzymology of the beta-oxidation pathway of valproate. Methods: Urine organic acids were analyzed by gas chromatography/mass spectrometry. In vitro studies were performed with heterologously expressed human 2-methyl-3-hydroxybutyryl-CoA dehydrogenase (MHBD) and fibroblasts from controls and a patient with MHBD deficiency using 2-methyl-3-hydroxybutyryl-CoA and 3-hydroxyvalproyl-CoA as substrates. The respective enzymatic activities were measured using optimized HPLC procedures. Short-chain enoyl-CoA hydratase (ECHS1) immunoprecipitation in a human liver homogenate was performed and hydratase activity was measured in the supernatants by HPLC, using crotonyl-CoA and Delta(2(E))-valproyl-CoA as substrates. Results: Patients on valproate therapy had a moderately increased urinary excretion of the isoleucine metabolite 2-methyl-3-hydroxybutyric acid. MHBD was found to convert 3-hydroxyvalproyl-CoA into 3-ketovalproyl-CoA. MHBD activity in control fibroblasts was comparable using both 2-methyl-3-hydroxybutyryl-CoA and 3-hydroxyvalproyl-CoA as substrates. In fibroblasts of a patient with MHBD deficiency, there was no detectable MHBD activity when 3-hydroxyvalproyl-CoA was used as substrate. Samples with immunoprecipitated crotonase had no detectable hydratase activity using both crotonyl-CoA and Delta(2(E))-valproyl-CoA as substrates. Discussion: This work demonstrates for the first time, that MHBD is the unique enzyme responsible for the dehydrogenation of 3-hydroxyvalproyl-CoA. Furthermore, we show that crotonase is the major, if not the single hydratase involved in VPA beta-oxidation, next to its role in isoleucine catabolism. (C) 2011 Elsevier Inc. All rights reserved.