The expression of H19 and insulin-like growth factor-II (IGF-II) genes is important for fetal growth, and the misexpression of these genes may also be involved in the development of some tumors. In human fetal adrenals, H19 and IGF-II expression levels are very high. We show here that H19 is strongly expressed (approximately 50% of the expression in fetal adrenals and 6-fold higher than that in adult liver) in normal adult adrenals (n = 9), adrenocortical adenomas (n = 28), and hyperplastic adrenals (n = 11). In four hormonally active adrenocortical carcinomas, very low levels of H19 ribonucleic acid (RNA) were detected, whereas IGF-II was highly expressed. In cultured adrenocortical cells, ACTH, (Bu)2cAMP, and cholera toxin increased H19 RNA accumulation 2- to 5-fold (P < 0.01), but had no significant effect on IGF-II messenger RNA levels. In pheochromocytomas (n = 22), H19 expression was variable, on the average, about 13% of the expression in the adjacent adrenal cortex. In primary cultures of pheochromocytoma cells, H19 RNA was not detectable via Northern blot analysis. Our data show that H19 expression is maintained at high levels in adult human adrenals and benign neoplasms. H19 RNA is up-regulated by ACTH in adult adrenocortical cells. The very low levels of H19 expression in hormonally active adrenocortical carcinomas suggest that loss of H19 expression may be associated with malignancy in these neoplasms.