Two highly purified low-molecular-weight (< 500 Da) Na-K-ATPase inhibitors, one originating from human plasma and the second from human urine, which both eluted in the identical locus from a C18 reversed-phase high-pressure liquid chromatography (HPLC) column, were compared with respect to (a) K effect on Na-K-ATPase inhibition; (b) displacement of [3H]ouabain from binding sites on purified hog brain Na-K-ATPase; (c) cross-reactivity with digoxin antibodies; and (d) vasoconstrictor effects in isolated rabbit femoral arteries. Inhibition of Na-K-ATPase by the plasma factor correlated inversely with K concentration, whereas inhibition by the urine factor correlated directly with K concentration. In the absence of K, the plasma factor displaced [3H]ouabain from both high- and low-affinity binding sites, whereas the urine factor displaced [3H]ouabain only from the low-affinity binding site. Neither factor possessed digoxin-like immunoreactivity. Both factors acted as direct vasoconstrictors, and potentiated the vasoconstrictor action of norepinephrine. The degree of vasoconstriction caused by the plasma factor diminished progressively with added K, indicating that the vasoconstrictor effect of this factor was mediated by the Na-K-ATPase pump. Thus, although both the plasma and urine Na-K-ATPase inhibitors are vasoconstrictors, their mechanisms of action are different.