Endogenous digitalis-like factors in humans are presumably cardenolides and bufadienolides. To test whether bufadienolide-like substances may circulate in human blood, we used antibodies from rabbits against the bufadienolide proscillaridin A to measure the concentration of cross-reacting material in human plasma with an indirect enzyme-linked immunosorbent assay. IgG had an apparent affinity of 2 x 10(-9) mol/L for proscillaridin A. It was specific for bufadienolides and did not cross-react with cardenolides or several steroid hormones. Extraction of human plasma with ethanol and fractionation of this extract over a high-performance liquid chromatographic reverse-phase C18 column with a propanol/isopropanol gradient resulted in the separation of three peaks of increasing hydrophobicity (ED1, ED2, ED3) that inhibited the sodium pump of human red blood cells and cross-reacted with proscillaridin A antibodies. The concentration of the proscillaridin A immunoreactivity ED1 in normotensive subjects had a geometric mean of 0.1 nmol/L, with a dispersion factor of 8.77. ED1 correlated positively in a group of 60 normotensive subjects, 22 patients with hypertension, and 19 patients with chronic renal failure with mean arterial blood pressure (log ED1 [nmol/L] = 0.013 x mm Hg-2.17, r = .25, P < .05), systolic pressure (log ED1 [nmol/L] = 0.010 x mm Hg-2.23, r = .32, P < .01), and pulse pressure (log ED1 [nmol/L] = 0.019 x mm Hg-1.80, r = .38, P < .0001). There was no correlation with other parameters of the donors. We conclude that several substances cross-reacting with proscillaridin A antibodies and inhibiting the sodium pump of human red blood cells circulate in human blood. The level of one of these substances (ED1) correlates with mean arterial and pulse pressures.