Cytochrome P-450C21 was successfully demonstrated in the human adrenal glands by a peroxidase-antiperoxidase method. All three cortical layers were stained in the normal adrenal glands, particularly the zonae glomerulosa and reticularis. Well-stained and faintly stained cells were intermingled in the zona fasciculata, suggestive of intrazonal variations. The immunoreactivity was particularly intense at the site of ACTH action, i.e., cells in micronodules and cells around myelolipomatous lesions in adrenocortical hyperplasia of Cushing's disease and sites of regeneration in the normal adrenal glands. In adrenocortical adenomas with Cushing's syndrome and primary aldosteronism, cells with large nuclei were generally stained well. In the adrenocortical tissue adjacent to a functioning adenoma, the immunoreactivity was observed only in the zona glomerulosa, especially in cases of primary aldosteronism. This finding is consistent with morphologic observations.