We have previously demonstrated that a 12 kD hypertension-associated protein (HAP) is elevated in essential hypertension and that this protein has the characteristics of natriuresis, inhibition of Na-K-ATPase, displaces 3H-ouabain from binding sites, and is vasoconstrictive in vitro. In the present study, plasma from 101 patients were examined [25 normals (N)<age 50, 13 N >age 50, 7 with acute congestive heart failure (CHF), 24 with chronic renal failure (CRF), on dialysis, 5 with idiopathic hyperaldosteronism (PA) and 27 with essential hypertension, untreated (EHT)]. Plasma was extracted with 32% acetonitrile, then analyzed by DELFIA for marinobufagenin and ouabain. In addition, from 32 patients (6 N <50, 6 N >50, 5 CHF, 5 CRF, 6 EHT, and 4 PA) SDS gradient gels were obtained. The 12 kD bands were extracted, analyzed for Na-K-ATPase inhibition, marinobufagenin and ouabain, and compared to 14 kD and 21 kD bands. Marinobufagenin was found to be elevated in CRF, EHT, PA and CHF. Ouabain was increased only in PA. When the relative optical densities of the 12 kD and 21 kD bands were contrasted, CRF, PA, and EHT were found to be increased and CHF to be decreased in the 12 kD band, with no discernible changes in the 21 kD bands. Following extraction of the bands, Na-K-ATPase inhibitory activity measured 38% in 18 pooled 12 kD bands, with essentially no activity found in the 14 kD or 21 kD bands. Thus only the 12 kD HAP band possessed all of the attributes of natriuretic hormone.