Eighteen linear analogues of [Arg8]vasopressin (AVP) were synthesized by systematically substituting the cysteine residues at positions 1 and 6 with a range of l-amino acids. Screening by competition ligand binding revealed that the combinations of amino acids residues tolerated at these positions was very restricted with respect to retention of vasopressin receptor (VPR) binding. Consequently, only three of the eighteen analogues investigated, [Pro1,Met6]AVP, [Gly1,Met6]AVP and [Phe1,Lys6]AVP, bound to the V1a receptor. Furthermore, these three peptides were all selective for the V1a receptor rather than the V1b, V2 and vasotocin receptors. In addition, although very homolous to the natural agonist, these analogues were in fact antagonists at V1a receptors. These data provide insights into the biophysical requirements at positions 1 and 6 of linear ligands for binding to V1a receptors and furthermore, supply clues to the nature of the receptor:ligand interaction.