Stereochemical aspects of the biotransformation of valproic acid (VPA) to four compounds believed to represent products of mitochondrial beta-oxidation, viz. delta 2(E)-VPA, delta 3-VPA, 3-hydroxy-VPA, and 3-oxo-VPA, were examined in freshly isolated rat hepatocytes. Following incubation of the individual enantiomers of [5-13C]VPA and analysis of products by GC/MS techniques, it was possible to determine for each metabolite the relative populations of molecules that had been formed by oxidation on the pro-R vs. the pro-S propyl group of the drug. Metabolism was found to exhibit a slight preference (approximately 1.3:1) for attack on the pro-S side-chain for all four compounds, consistent with the hypothesis that this group shares a common metabolic origin. In contrast, the hepatotoxic terminal olefin, delta 4-VPA, was formed with marked enantiotopic differentiation (approximately 3.8:1) favoring the pro-R side-chain. The reason for the surprisingly low stereo-selectivity displayed by the products of beta-oxidation was investigated with the aid of [3-2H] delta 2(E)-VPA as metabolic substrate. Following incubation with rat hepatocytes, 35% of the substrate remaining after 2 hr was found to have been isomerized to [3'-2H] delta 2(E)-VPA. Because delta 2(E)-VPA is known to be formed from VPA-CoA through the action of 2-methyl-branched-chain acyl-CoA dehydrogenase, it is proposed that the three-carbon side-chains of both parent drug and delta 2(E)-VPA are interconverted as a consequence of reversibility in the second half-reaction of this enzymatic process.