A Na+,K(+)-ATPase inhibitor was purified from 88.6 l pig urine with a yield of approximately 10 micrograms. It inhibits the ouabain-sensitive uptake of 86Rb into human erythrocytes and the specific binding of ouabain to Na+,K(+)-ATPase. It also exhibits cross-reactivity to anti-ouabain serum. The purification procedure consisted of adsorption chromatography on an Amberlite XAD-2 column, preparative scale C18 low-pressure liquid chromatography (LPLC), and five steps of HPLC with five different types of reverse-phase columns. The dose dependence of the purified substance for the inhibition of ouabain-sensitive Na+,K(+)-ATPase activity and 86Rb uptake into human erythrocytes, and for the ouabain-displacing activity, paralleled those of ouabain, spanning two orders of magnitude in concentration range. However, the curve obtained from the cross-immunoreactivity with anti-ouabain serum did not parallel that of ouabain. The inhibitory potencies of the purified substance against the Na(+)-pump and ouabain-binding were diminished with increasing K+ concentration, exhibiting characteristics typical of cardiac glycosides. This substance had no effect on Ca(2+)-ATPase activity in human erythrocyte plasma membrane and skeletal-muscle sarcoplasmic reticulum, nor on Mg(2+)-ATPase activity. Acid treatment with 6 M HCl at 115 degrees C for less than 1 min destroyed approximately 82% of the inhibitory activity of the purified substance against Na(+)-pump activity. Alkaline treatment with 0.2 M NaOH at 23 degrees C for 2 h and heat treatment at 150 degrees C for 30 min partially destroyed the inhibitory activity. Boiling for 10 min and digestion by various enzymes did not affect the activity. Molecular mass was estimated to be 620 Da by gel-filtration column chromatography. Preliminary MS analysis suggested that the purified substance has a molecular mass of 625 Da. An 1H-NMR study revealed that this substance does not contain a tertiary methyl group. The results suggest that the purified Na+,K(+)-ATPase inhibitor is not a peptide and is distinct from any of the known cardiotonic steroids or various substances previously reported to exhibit Na+,K(+)-ATPase inhibitory activity. Thus, the purified substance may be a novel endogenous regulator of Na+,K(+)-ATPase.