The molecular mechanisms regulating adrenal glomerulosa production of aldosterone remain poorly defined in part due to limitations in obtaining sufficient adrenal glomerulosa tissue and cells. The only current adrenocortical cell model for aldosterone studies is the NCI‐H295 line, which was developed from a human adrenocortical carcinoma (HAC). Herein, we attempted a similar approach with the goal of isolation and characterization of clonal cell lines from a HAC. To determine if the HAC cells represent a model for adrenal glomerulosa, the cells were treated with angiotensin II (Ang II) and potassium (K+), followed by examination of aldosterone production and steroidogenic enzyme mRNA expression. Treatment with Ang II (10 nM) and K+ (18 mM) increased aldosterone production in a time‐dependent manner. Ang II and K+ increased expression of mRNA encoding all enzymes needed for aldosterone synthesis; namely StAR, CYP11A, HSD3B2, CYP21, and CYP11B2. In addition, the cells expressed mRNA for the Ang II type 1 receptor and its expression was increased upon treatment with Ang II and K+. The current study describes the development of a novel human adrenal cell line with responses seen in normal glomerulosa cells. The HAC cell line should provide an important alternative model system for defining the molecular mechanisms regulating aldosterone production.