Excessive secretion of aldosterone from the adrenal results in the most common form of endocrine hypertension. An understanding of the regulatory processes involved in aldosterone synthesis and release is needed to define the biomolecular mechanisms controlling excessive production of aldosterone. However, in vitro studies regarding the regulatory mechanisms of human aldosterone production have been limited because of difficulties in obtaining tissue and the subsequent isolation of aldosterone-secreting glomerulosa cells. Herein we describe an adrenocortical carcinoma cell line, NCI-H295, which provides a suitable angiotensin-II (AII)-responsive model system to investigate the acute and chronic regulation of aldosterone synthesis. The cells were characterized with regard to the effects of AII on second messenger systems, aldosterone release, and levels of aldosterone synthase (P450c18) mRNA. In the presence of lithium, AII caused a rapid, but transient, increase in the production of inositol tris- and bisphosphates, whereas a prolonged gradual accumulation of inositol monophosphate occurred. Treatment with AII resulted in a 4.5-fold increase in total inositol phosphates in a concentration-dependent manner and an increase in intracellular cytoplasmic free Ca2+. Significant increases in aldosterone (3.5-fold) were detected within 1 h of AII addition. Aldosterone release occurred in a concentration-and time-dependent manner. The type 1 AII (AT1) receptor was shown to be responsible for activation of phosphoinositidase-C, increased intracellular free Ca2+, and aldosterone production, as determined by use of the AT1 receptor antagonist DuP753. In addition, AII treatment resulted in a time-dependent increase in levels of P450c18 mRNA, as detected by RNAse protection assay. In summary, NCI-H295 cells provide a valuable model system to define mechanisms regulating human aldosterone production.