Furanoid 8-epidiosbulbin E acetate (EEA) is a major constituent of herbal medicine Dioscorea bulbifera L. (DB), a traditional Chinese medicine herb. Our preliminary studies demonstrated that administration of EEA caused acute hepatotoxicity in mice, and the observed toxicity required cytochromes P450-mediated metabolism. Metabolic activation studies of EEA were performed in vitro and in vivo. Microsomal incubations of EEA supplemented with N-acetyl lysine (NAL) and glutathione (GSH) generated six metabolites (M1-M6). M1-M4 were characterized as pyrrole derivatives, and M5 and M6 were pyrrolinones. M2-M6 were detected in bile and/or urine of rats given EEA. Dimethyldioxirane-mediated oxidation of EEA in the presence of NAL and GSH produced M1-M6, all of which were generated in microsomal incubations. The structures of M3 and M6 were confirmed by (1)H and (13)C NMR. These findings provide evidence for the metabolic activation of EEA to the corresponding cis-enedial intermediate both in vitro and in vivo. Ketoconazole inhibited the microsomal production of the cis-enedial, and P450 3A4 was found to be the primary enzyme involved in the bioactivation of EEA.