The role of an endogenous sodium pump inhibitor in the pathogenesis of hypertension has been reported in several papers. Unfortunately, because of the unknown structure and lack of biochemical characterization, some discrepancies have arisen. In this study we report a method to obtain extracts from human plasma that are able to inhibit Na+/K+-ATPase in vitro. Preliminary characterization was carried out, which showed that the extracts are able to inhibit Na+/K+-ATPase, pNPPase, and [3H]ouabain binding to red blood cells. The enzyme inhibition is not due to vanadate, FFA, or bivalent cations, and it seems to be reversible, dose-dependent, and largely prevented in E1 conformation of the enzyme. These results seem to support the hypothesis that human plasma contains a sodium pump inhibitor with many characteristics of a "ouabainlike" compound.