Valproic acid (VPA) is a drug known for idiosyncratic hepatotoxicity and is associated with oxidative stress. It is metabolized extensively with at least one pathway leading to reactive metabolites. The primary aim of the present study was to determine whether oxidative metabolites of VPA generated in situ contribute to the toxicity of the parent drug in sandwich-cultured rat hepatocytes. Concentration-response experiments with VPA produced median effective concentration values (mean ± SEM) of 1.1 ± 0.4, 12.2 ± 1.4, and 12.3 ± 1.9mM in the 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-1; cell viability), lactate dehydrogenase (LDH; necrosis), and 2',7'-dichlorofluorescein (DCF; oxidative stress) assays, respectively. At equimolar concentrations, only the unsaturated metabolites of VPA gave responses comparable to VPA, with 2,4-diene-VPA calculated to be 3-, 6-, and 10-fold more potent than VPA in the WST-1, LDH, and DCF assays, respectively. In support of a role for reactive metabolites, 2-fluoro-2-propylpentanoic acid, which is relatively resistant to biotransformation to form a 2,4-diene metabolite, yielded little or no toxicity when compared with the nonhepatotoxic octanoic acid or the vehicle-treated control. By comparison, attenuating the in situ formation of 2-propylpent-4-enoic acid (4-ene-VPA), 3-hydroxy-2-propylpentanoic acid, 4-hydroxy-2-propylpentanoic acid, and 5-hydroxy-2-propylpentanoic acid by an inhibitor of cytochrome P450 (1-aminobenzotriazole) did not alter the effects of VPA on the WST-1, LDH, or DCF assay. Overall, VPA toxicity in sandwich-cultured rat hepatocytes is independent of the in situ formation of cytochrome P450-dependent oxidative metabolites, including 4-ene-VPA. However, the data obtained from structural analogues of VPA suggest that biotransformation does appear to play a role in VPA toxicity in rat hepatocytes.