Ouabain is a specific inhibitor of the sodium pump. This steroid has been found in the mammalian circulation in significant amounts and may be of adrenal origin. Secretion of ouabain from adrenal cells has been little studied and the purpose of the present work was to determine the adrenal distribution of ouabain, aldosterone and cortisol, and to characterize the effects of ACTH and angiotensin II on the secretion of these steroids in primary cultures of bovine adrenocortical cells. In fresh bovine adrenals, the cortical to medullary ratios for aldosterone, cortisol and ouabain were 14, 4.25 and 2.5, respectively. All three steroids were detected in elevated amounts in the conditioned medium of primary cultures of adrenocortical cells. Reverse phase HPLC of the secreted ouabain immunoreactivity showed it was isopolar with commercial ouabain. In the presence of 10 nM ACTH or angiotensin II, the secretion of all three steroids increased significantly with similar time courses. The stimulated secretion of ouabain exceeded the intracellular content of this steroid in either control or activated cells by 3-5 fold. The amount of angiotensin II stimulated ouabain secretion was greater from cells incubated in larger volumes. These results show that ouabain is enriched in the bovine adrenal cortex, and is secreted by primary cultures of these cells. The secretion of ouabain is increased by ACTH and angiotensin II, is due to either de novo synthesis or transformation of an intracellular precursor that is not overtly immunoreactive, and is feedback regulated by either ouabain itself or a cosecreted factor. These cells may be useful to study stimulus-secretion coupling and the biosynthetic pathway of ouabain.