Ouabain or strophantin-g is a widely used cardiac glycoside acting by the inhibition of the membrane-bound enzyme Na⁺-K⁺-ATPase. Hamlyn et al. showed in 1991 that the endogenous counterpart of this glycoside is ouabain itself (or a closely related isomer of it). They showed that cultured bovine adrenocoticol cells produce ouabain. The aim of our group was to study the regulation of the production of ouabain-like factor (OLF) in rat adrenal cortex. Zona glomerulosa and fasciculate tissue slices and cells were used. Our results show that both parts of the adrenal certex produce ouabain-like factor and that ACTH and angiotensis-ll increase ouabain secretion dose-dependently. Fine changes in extracellular K⁺ concentration, which is an important regulator of aldosterone secretion, influenced ouabain secretion, too. Human adrenals (normal and adenomatous) were also investigated. Our results show that OLC production is regulated by high potassium concentration and this regulation is different in normal and high aldosterone producing adrenocortical cells. It has been recognised long ago that the regulation of aldosteroe secretion is multifactorial (ACTH, angiotensin-ll, atrial natriuretic peptide, potassium). Among the numerous paracrine regulators now OLF has been emerged as a new factor. At concentrations of 10^-5 M or 10^-4 M, ouabain has a stimulatory effect on basal aldosterone production; whereas at concentrations of 10^-3 M, it has either no effect or inhibitory effects. We have shown previously in the rat that chronic treatment with ouabain increases aldosterone production without changing renal renin levels. In rat adrenocortical tissue slices and cell preparations, the effects of ouabain on aldosterone production strongly depended on its concentration and the K⁺ concentration of the medium. We demonstrated that there is an interation between the effect of ouabain, atrial natriuretic peptide, angiotensin-ll and potassium on aldosterone production by collagenase dispersed rat zona glomerulosa cells. This may be of physiological significance in the regulation of aldosterone secretion.