The mechanism(s) of the "aldosterone turn-off phenomenon", hypoaldosteronemia following chronic ACTH administration, remains unclear. Our previous observation that antioxidants prevented turn-off prompted us to evaluate the chronic effect of ACTH on the enzymatic antioxidant system as well as P450aldo activity and expression of CYP11B2 in adrenal zona glomerulosa. Male Wistar rats were administered ACTH-Z for 5 days with or without antioxidants, vitamin E or DMSO. Adrenal capsules were prepared for P450aldo activity measurement and mRNA content determination by competitive RT-PCR, and immunoreactivity of Mn-SOD in whole adrenals was evaluated. ACTH decreased the P450aldo activity and mRNA level of CYP11B2 in adrenal capsules, while co-administration of vitamin E or DMSO partially blocked this inhibition. ACTH increased Mn-SOD mRNA and immunoreactivity but decreased GPx mRNA. These results suggest that prolonged ACTH treatment increases oxidative stress in the zona glomerulosa and an imbalance in the ratio of Mn-SOD to GPx, possibly via corticosterone overproduction in the zona fasciculata, resulting in the downregulation of CYP11B2. Vitamin E and DMSO might thus protect CYP11B2 expression through their antioxidant actions.