The functional characterization of putative endogenous inhibitors of the Na(+)-K(+)-ATPase has been greatly hindered by spare amounts extractable from biological sources. We therefore used a miniaturized, two-sided test system consisting of ATP-filled liposomes containing dispersed, randomly oriented renal Na(+)-K(+)-ATPase molecules to study effects of a low-molecular-weight, nonpeptidic Na(+)-K(+)-ATPase inhibitor extracted from bovine hypothalamus. With this test system, Na(+)-K(+)-ATPase inhibition produced by a single dose of 0.1 U (congruent to 75 fmol) of the hypothalamic inhibitory factor (HIF) as well as the membrane permeation of a single unit (approximately equal to 750 fmol) became measurable, and an estimation of the minimal number of HIF molecules per unit could be made. By a molecular mechanism involving positive cooperativity, HIF potently and completely blocked active 86Rb+ transport catalyzed by the right-side-out-oriented pump population, with an average 50% inhibitory concentration of 3.5 x 10(-8) M, indicating a roughly 30-fold higher apparent affinity than ouabain. By studying inhibition of the inside-out-oriented pump population, comparison of the membrane permeability of HIF to that of various cardiac glycosides of known hydrophobicity further indicated that HIF is not entirely ouabain-like as HIF penetrates the liposomal membrane, whereas ouabain does not. Besides the cardiac glycosides, HIF is the only compound tested thus far in the purified system that displays such striking transport inhibition. Other known or proposed endogenous Na(+)-K(+)-ATPase inhibitors, including unsaturated fatty acids, palytoxin, dehydroepiandrosterone, and vanadate, produce only partial transport inhibition even at high concentration.