We were able to purify two distinct sodium pump inhibitors to homogeneity from human urine based on [3H]ouabain-displacing activity from intact human erythrocytes. The polar and less polar compounds were eluted off the C18 reverse-phase column with 18% and 31% acetonitrile, respectively. The polar compound cross-reacted very weakly with specific antidigoxin antibody and lacked a characteristic ultraviolet absorption peak between 190 and 300 nm. The less polar compound showed a prominent digoxinlike immunoreactivity and had an ultraviolet spectrum similar to that of digoxin. We examined the effects of these compounds on cytosolic free calcium concentration in cultured rat vascular smooth muscle cells (A10 cells) using the fluorescent calcium chelator fura-2. Only the polar ouabain-displacing compound caused a significant increase, from 108 +/- 7 to 162 +/- 8 nM (n = 6, p less than 0.01), in cytosolic free calcium concentration in A10 cells. The rise in cytosolic free calcium concentration induced by the polar ouabain-displacing compound tended to be slower in onset and more sustained than that induced by arginine vasopressin. In contrast, ouabain and bufalin had no appreciable effects on cytosolic free calcium concentration in A10 cells. These results suggest that the polar ouabain-displacing compound we isolated from human urine may possess a vasoactive property and may play an important role in the modulation of vascular tone.