Aldosterone-producing adenoma (APA) is a major cause of primary aldosteronism, leading to secondary hypertension. Somatic mutations in the gene for the α1 subunit of the Na+/K+-ATPase were found in about 6% of APAs. APA-related α1 subunit of the Na+/K+-ATPase mutations lead to a loss of the pump function of the Na+/K+-ATPase, which is believed to result in membrane depolarization and Ca2+-dependent stimulation of aldosterone synthesis in adrenal cells. In addition, H+ and Na+ leak currents via the mutant Na+/K+-ATPase were suggested to contribute to the phenotype. The aim of this study was to investigate the cellular pathophysiology of adenoma-associated Na+/K+-ATPase mutants (L104R, V332G, G99R) in adrenocortical NCI-H295R cells. The expression of these Na+/K+-ATPase mutants depolarized adrenal cells and stimulated aldosterone secretion. However, an increase of basal cytosolic Ca2+ levels in Na+/K+-ATPase mutant cells was not detectable, and stimulation with high extracellular K+ hardly increased Ca2+ levels in cells expressing L104R and V332G mutant Na+/K+-ATPase. Cytosolic pH measurements revealed an acidification of L104R and V332G mutant cells, despite an increased activity of the Na+/H+ exchanger. The possible contribution of cellular acidification to the hypersecretion of aldosterone was supported by the observation that aldosterone secretion of normal adrenocortical cells was stimulated by acetate-induced acidification. Taken together, mutations of the Na+/K+-ATPase depolarize adrenocortical cells, disturb the K+ sensitivity, and lower intracellular pH but, surprisingly, do not induce an overt increase of intracellular Ca2+. Probably, the autonomous aldosterone secretion is caused by the concerted action of several pathological signaling pathways and incomplete cellular compensation.