Cytochrome P-450 specific for steroid 17 alpha-hydroxylation (P-45017 alpha) was immunolocalized in normal and hyperfunctioning adrenal glands of pigs, bovines, and humans, using a specific IgG fraction raised against the enzyme. P-45017 alpha was present in the zona fasciculata (ZF) and zona reticularis (ZR), but not in the zona glomerulosa (ZG), in pig, bovine, and human adrenal glands. In the adrenal glands of patients with Cushing's disease, the positive immunoreactivity to P-45017 alpha was intense in ZF and ZR, particularly in cortical micronodules, corresponding to the sites of active steroidogenesis. Cells of hyperplastic ZG and outer ZF in the adrenal glands of idiopathic hyperaldosteronism were negative for P-45017 alpha. In aldosteronoma, positive immunoreactivity was observed in some tumor cells, which is consistent with cortisol production and its responsiveness to ACTH in aldosteronoma. In the attached adrenal glands of aldosteronoma, the immunoreactive P-45017 alpha was clearly present in the inner ZF and ZR, suggesting persistent androgen production. In Cushing's adenoma, the positive immunoreactivity was intense in tumor cells, and the ZR of the attached adrenal glands was weakly immunoreactive.