Although polychlorinated biphenyls (PCBs) have been shown to accumulate in the adrenal, little is known about the effects of these endocrine disruptors on adrenal steroidogenesis. Our previous studies showed that high concentrations of PCB126 stimulated CYP11B1 and CYP11B2 mRNA expression and consequently raised cortisol and aldosterone synthesis in the human adrenocortical H295R cells, respectively. In this study, we further investigated the mechanism underlying the PCB126-induced steroidogenic alterations. We first examined the role of the PCB126 nuclear receptor AhR using a potent antagonist 3',4'-dimethoxyflavone (3',4'-DMF). Although 3',4'-DMF abolished AhR-dependent transcriptional activity, it could not block PCB126-stimulated CYP11B1 and CYP11B2 induction. Oppositely, 3',4'-DMF synergistically increased the stimulatory effects of PCB126. Furthermore, PCB39, 77, 132, 156 and 169, no matter being AhR ligands or not, all could increase CYP11B1 and CYP11B2 mRNA accumulation. Promoter analyses demonstrated that PCB126 had little effects on the transcription rate of both genes, while RNA degradation assays showed that PCB126 protected both transcripts from degradation. In contrast, 3',4'-DMF exhibited positive effects on transcription, but no influence on transcript stability. The synergistic induction of CYP11B1 and CYP11B2 mRNA levels by the PCB126/3',4'-DMF cotreatment might result from the combination of transcriptional regulation by 3',4'-DMF and post-transcriptional regulation by PCB126. This study also demonstrated that an internal region of CYP11B1 mRNA (nucleotide 881-1285) was important for PCB126-mediated transcript stabilization. From these findings, we concluded that PCB126 upregulated steroidogenic CYP11B1 and CYP11B2 mRNA expression not via AhR-mediated transcriptional activation, but by increasing post-transcriptional mRNA stability.