In order to study the influence of the hypothalamic-pituitary-adrenal axis on the levels of endogenous digitalis-like substances (EDLS) in plasma and urine, eight healthy subjects (25–40 years old) were given dexamethasone 1 mg orally and tetracosactide (an ACTH analog) 0.25 mg iv, on separate occasions. The circulating levels of EDLS, TSH, PRL and AVP following administration of either test drug, and under control conditions, were measured by a RIA for digoxin and specific RIAs for each hormone. Plasma cortisol was measured by liquid chromatography. The area under the curve (AUC) of hormone levels between 08.00 and 09.30 was used for data comparisons. Urine was collected before and after each test dose, and analysed for cortisol levels by gas chromatography/mass spectrometry, and for digitalis-like activity both by RIA and by a bioassay measuring 86Rb-uptake into red blood cells. Dexamethasone suppressed the AUC of plasma and urine levels of cortisol (p=0.0001 and p<0.01, respectively) and immunoreactive EDLS (p=0.0007 and p<0.01), as well as serum levels of TSH (p=0.0002) and PRL (p=0.001), but did not alter AVP levels. The biological digitalis-like activity in the urine measured by the 86Rb-uptake assay was decreased, but not to a statistically significant degree. ACTH increased the levels of cortisol in plasma (p=0.0001) and urine (p<0.01) and the immunoreactive EDLS in plasma (p = 0.03), but not in urine. There were no effects of ACTH on TSH, PRL or AVP. There are alternative explanations for the discrepancy between the effects on EDLS levels in plasma and urine: methodological difficulties in quantitating EDLS, the doses of dexamethasone and ACTH used for the adrenal function tests, and that other hypothalamic or pituitary factors than ACTH may contribute to a significant degree in the regulation of EDLS levels. Taken together, the results of the present study support the hypothesis that EDLS is of adrenal origin, rather than hypothalamic or pituitary.