We investigated the presence of endogenous Na-K-ATPase inhibitor(s), ie, ouabain-like factors (OLFs), in the urine of salt-loaded healthy subjects. For this purpose 24-h urine was collected on days 3, 4, and 5 of high sodium intake (> 30 g NaCl/day). The samples then were lyophilized. Redissolved urine concentrates were acidified (pH 3.5) and subjected to gelchromatography on a Sephadex G-25 column where the OLFs eluted in the post-salt fraction IV. When lyophilized fraction IV was rechromatographed on Sephadex G-10, OLFs with molecular mass (M(r) of approximately 400 eluted in a late fraction IV/8 separate from added ouabain, ouabagenin (or digoxin), which eluted shortly after void volume. With the subsequent reverse-phase HPLC of fraction IV/8 a polar OLF-1 eluted in fraction IV/8a after the void volume in the water phase and a more apolar OLF-2 eluted at 20% acetonitrile in fraction IV/8d. Only the more apolar OLF-2 cross-reacted with a digoxin antibody. By preparative thin-layer chromatography OLF-1 and OLF-2 were purified as single compounds with potent dose-dependent Na-K-ATPase inhibition and Ki-values approximating 1.5 x 10(-5) mol/L and 1.5 x 10(-4) mol/L, respectively. Mass-spectroscopy (MS) showed M(r) of 391 and 1H-NMR characterized the endogenous urinary apolar OLF-2 as a compound that is structurally totally unrelated to ouabain; infrared (IR) spectroscopy of OLF-1 and OLF-2 also revealed no similarity with ouabain.(ABSTRACT TRUNCATED AT 250 WORDS)