Polybrominated diphenyl ethers (PBDEs) are additive flame retardants that have been found in the environment as well as human tissues. Environmental concentrations of these compounds have been increasing in many parts of the world in recent years. Due to their structural similarity, PBDEs are believed to have similar toxicity to PCBs, but their toxicological properties are still being determined. In this study, the steroidogenic effects of hydroxylated, methoxylated and/or chlorinated derivatives of PBDEs were assessed at both the gene and enzyme/hormone levels in the H295R human adrenocortical carcinoma cell line. The expression levels of 10 steroidogenic genes were measured using quantitative real-time PCR (Q-RT-PCR). Aromatase activity in the cells and sex steroid (testosterone (T) and 17beta-estradiol (E2)) concentrations in the culture medium were also measured. CYP11B2, which regulates the synthesis of aldosterone, was the most sensitive gene and was induced by most of the compounds tested in this study. CYP19 gene expression, aromatase activity, and E2 production were also affected by several metabolites, but no consistent relationship was observed between these endpoints. Several PBDE metabolites showed some potential ability to interfere with steroidogenesis, including 5-Cl-6-OH-BDE-47, a biologically relevant BDE-47 metabolite, which significantly decreased aromatase activity and E2 production at a concentration of 10 microM. The results of this study indicate that PBDE metabolites affect steroidogenesis in vitro and that they may have the potential to affect steroidogenesis and reproduction in whole organisms.