Vasopressin (AVP) and oxytocin (OT) are two nonapeptides differing at position 3, in the cyclic part of the peptide, and at position 8, in the C-terminal tripeptide. In this study, we have evaluated the interactions between these two positions of the hormones and the oxytocin receptor (OTR), the V1a and the V2 vasopressin receptors. The contribution of these two positions to receptor selectivity was analyzed by using several peptide analogues bearing substitutions at either position 3 or 8. The putative interactions between receptor residues and hormone residues at position 3 and 8 were then deduced by using a three dimensional model of the neurohypophysial hormones docked into their respective receptors. On the basis of this model, we found that the lateral chain of residue 8 might interact with residues located in the first extracellular loop. By using site-directed mutagenesis on the cloned receptors, we identified a non-conserved residue in the first extracellular loop that interacts with the lateral chain of residue 8 in the hormone. We demonstrated that this interaction is crucial for receptor selectivity to the different agonists.