Immunohistochemical analysis of steroidogenic enzymes (P-450 side-chain cleavage, 3 beta-hydroxysteroid dehydrogenase, P-450 C21-hydroxylase, P-450 17 alpha-hydroxylase and P-450 11 beta-hydroxylase) was performed on fifteen cases of small adrenocortical adenomas, which were detected incidentally in hormonally asymptomatic patients (non-functioning adrenocortical tumor), in order to study steroidogenesis in these tumors. Immunolocalization revealed that all cases examined expressed all the enzymes in the adrenocortical steroidogenic pathway to various degrees, and in twelve cases abnormalities of precursor hormones and steroid metabolites were clinically observed. Attached non-neoplastic adrenals were present in twelve cases. Among these twelve cases, eight showed cortical atrophy, especially in the zona fasciculata. These atrophied adrenals expressed little immunoreactivity of the enzymes examined. These results strongly indicate that most of small non-functioning adrenocortical tumors have the capacity to produce biologically active steroids including cortisol, although not necessarily associated with hypercorticism. Especially in the cases with cortical atrophy in attached non-neoplastic adrenals, it is considered that autonomous neoplastic production and secretion of cortisol may be insufficient to cause clinical and routine laboratory abnormalities but sufficient to subtly alter the hypothalamic-pituitary-adrenal axis by suppressing ACTH and/or CRF secretion and to result in adrenocortical atrophy. It should be recognized that corticosteroidogenesis does take place in the majority of clinically small non-functioning adrenocortical tumors incidentally detected in hormonally asymptomatic patients when managing these patients.