Endothelins (ET) are 21-amino acid peptides that bind to membrane receptors to initiate a wide range of pathophysiological effects. PD-156707, L-749329, Ro-470203, and A-127722 are potent non-peptide ET receptor antagonists developed recently. When tested in human and rat plasma, both ET-1 and -3 and the four aforementioned antagonists exhibited a high degree (>98%) of plasma protein binding. When ET-1 binding to the receptors was examined, 5% (v/v) of human plasma inhibited ET-1 binding to both ET_a Mid ET_B receptors by 80 – 90%. Similarly, 5% (w/v) of human serum albumin inhibited ET-1 binding by 82%, suggesting that the major protein component in plasma which interfered with ET-1 binding to the receptors was serum albumin. Competition studies show that, in the absence of human serum albumin, the IC₅₀ values of PD-156707, L-749329, Ro47-0203, and A-127722 were 0.37, 0.29, 5.7, and 0.22 nM, respectively. Addition of increasing doses of human serum albumin incrementally decreased the potency of the antagonists; in the presence of 5% of human serum albumin, the IC₅₀ values increased to 62.8, 50.2, 122.7, and 6.72 nM for PD-156707, L-749329, Ro-470203, and A-127722, respectively. In conclusion, ET and ET receptor antagonists exhibit a high degree of binding to plasma proteins, especially serum albumin. Consequently, serum albumin inhibits ET binding to its receptors, and also decreases the potency of ET receptor antagonists. Our findings may explain the discrepancy observed for ET receptor antagonists between in vitro and in vivo potencies.