Objective: To investigate the metabolites of polybrominated diphenyl ether 99 (BDE-99) and its related cytochrome P450s in an in vitro system. Methods: Rat primary hepatocytes were isolated and treated with BDE-99 for 24-72 h. Metabolites were then extracted from the hepatocytes and media, and detected by GC/MS. Several mRNAs of metabolic enzymes were also extracted from the same cells and the gene expression levels were determined using quantitative real-time PCR. In addition, selected recombinant cytochrome P450s (CYPs) were expressed in a bacurovirus/sf9 system, and these were further used to explore the metabolism of BDE-99 in vitro. The parent depletion approach was used for screening the ability of CYPs to eliminate BDE-99. Results: A reductively debrominated metabolite, BDE-47, and three oxidative metabolites, 2, 4, 5-tribromophenol, 5-OH-BDE-47, and 5'-OH-BDE-99, were identified from the BDE-99-treated rat hepatocytes, whereas no MeO metabolite was detected in the system. RT-PCR analysis showed that CYP 3A23/3A1, 1A2, and 2B1/2 were induced by BDE-99. Furthermore, using the heterological expressed CYP proteins in in vitro BDE-99 metabolism experiments we found that CYP1A2 and CYP3A4 showed the highest metabolic efficiency for BDE-99, with the metabolic clearance rates of CYP1A2 and CYP3A4 being 30.3% and 27.7%, respectively. CYP1A1 and CYP2A6 displayed relatively low clearance rates, while CYP2E1 seemed not to be associated with the BDE-99 metabolism. Conclusions: In our in vitro rat primary hepatocyte metabolism system, four metabolites of BDE-99 were identified, and CYP3A4 and CYP1A2 were demonstrated to be involved in the BDE-99 metabolism.