It is well known that ascorbic acid (Asc) is highly concentrated in the adrenal gland, but its function in the gland is not thoroughly elucidated. We therefore examined the possibility that Asc participates in steroidogenic monooxygenase systems of the adrenal cortex with the aid of the regenerating system including outer mitochondrial membrane cytochrome b (OMb). When Asc availability was limited in rat mutants unable to synthesize Asc, the increase in plasma aldosterone concentration under Na-deficiency was suppressed without effect on plasma corticosterone concentration. Aldosterone formation in the isolated mitochondrial fraction of the zona glomerulosa (zG) of the adrenal cortex was stimulated by the addition of Asc and NADH, while corticosterone formation was not. Consistently zG showed a high level of Asc regeneration activity and was rich in OMb among adrenocortical zones. Taken together, the enhanced aldosterone formation that is catalyzed by one of the steroidogenic monooxygenases, P450aldo, may be supported by Asc with its regenerating system.