As part of a program to design potent antidiuretic vasopressin antagonists and to define the minimum effective pharmacophore requirements for vasopressin (VP) antagonist activity, we studied the importance of the C-terminal tripeptide of a previously reported peptide antagonist of arginine-vasopressin (AVP,1). The proline residue at position 7 in AVP is proposed to impart a conformational constraint to the peptide backbone that is essential for V2-receptor agonist activity. Since the structure-activity relationships for VP agonists and antagonists are different, we investigated the effect of proline on antagonist activity, by synthesizing analogue 3 lacking this residue. This analogue was found to retain a high degree of antidiuretic antagonist activity. Since deletion of the Gly residue at position 9 of the antagonist did not adversely affect VP antagonist potency, several vasopressin antagonist analogues (4-7 and 9) that lacked both the Pro and Gly residues were also studied. These, too, were found to block vasopressin V2-receptor activity. Our results indicate that neither the proline nor glycine residues are essential for antagonism of the V2 receptor.