An endogenous inhibitor of Na+, K+-ATPase was extracted from human plasma and sera and concentrated by a novel reverse-phase octadecylsilane chromatography method. The active extracts (eluates) were dried and reconstituted in the minimum volume of the non-adsorbed fraction of the plasma from which they had been derived. Reconstituted eluates, non-adsorbed plasma fractions and native plasma samples were then tested for their ability to inhibit phosphate production in standard Na+, K+-ATPase incubation mixtures. In a pilot study 31 samples of pooled normal human sera were assayed. The eluates gave a significantly lower production of phosphate than the non-adsorbed fractions or the native sera (n = 31, p less than 0.0025). Further concentration of the eluates by repeated chromatography increased the inhibitory power of the eluate proportional to the concentration achieved, as quantified by ouabain dose-equivalents. In clinical studies, samples from 12 normotensive subjects and from 12 untreated patients with essential hypertension were tested. Significant inhibition of the ATPase by the eluates, as compared to the corresponding non-adsorbed fractions was seen for samples from both normotensive (p less than 0.05) and hypertensive (p less than 0.05) subjects. There was no significant difference in incidence or degree of inhibition between the normotensive and hypertensive groups. This study provides direct evidence for the presence of an endogenous inhibitor of Na+, K+-ATPase in human plasma.