Outer mitochondrial membrane cytochrome b5 (OMb) originally found in rat liver is an isoform of cytochrome b5 (b5) of the endoplasmic reticulum. In contrast to accumulated data on the physiological roles of b5, functions of OMb have not been well characterized except for its involvement in regeneration of ascorbic acid [i.e., in a semidehydroascorbate reductase (SDAR) system]. By using highly specific antibodies against rat OMb, we found immunohistochemically that OMb in the rat adrenal gland was most abundant in the zona glomerulosa (zG) among the three cortical zones, and the expression level was enhanced on angiotensin II-stimulation. SDAR activity was found in zG and inhibited by anti-OMb antibody. Moreover, the increase in plasma aldosterone concentration under Na+ -deficiency was suppressed by limited ascorbic acid (Asc) availability in rat mutants unable to synthesize Asc, while plasma corticosterone concentration was not affected. These data suggest that OMb, present abundantly in zG, participates in aldosterone formation in zG of rat under angiotensin II-stimulation through regeneration of Asc.