Prolactin-releasing peptides (PrRPs) are two novel bioactive peptides of 20 and 31 residues, dubbed respectively PrRP20 and PrRP31, isolated from bovine hypothalamic tissues as ligands of the orphan seven-transmembrane domain receptor Hgr3. The first biological activity identified for these peptides was the release of prolactin. Recent data on biological activities of PrRPs as well as on the localization of their receptors in numerous central nervous system sites suggested new potential actions of PrRPs in the regulation of the central nervous system and the possibility of identifying an alternative central role for these peptides. We describe here the synthesis and the structural characterization of the peptide PrRP20 by CD and NMR spectroscopies. A 3D model was built on the basis of the NMR data collected in a water/sodium dodecyl sulfate mixture. This system provides an amphipatic medium able to mimic the cell membrane. The main structural feature of the PrRP20 is an alpha-helical secondary structure spanning the 10 C-terminal residues. The conformational properties of PrRP20 are discussed in considering the sequence similarity observed between the Hgr3 and the neuropeptide Y (NPY) receptors. This similarity, together with the data showing a number of biological activities common to PrRP and NPY peptides, leads us to formulate the hypothesis that similar structural elements could exist in the ligands as well. In fact, PrRP20 and NPY are well aligned in the C-terminal portion, where they share an amphipatic alpha-helical secondary structure. Interestingly, the homology between the two sequences involves residues crucial for NPY biological activity. The conformational characterization of PrRP20 and the comparison with NPY are a valuable starting point for the rational design of subsequent SAR studies aimed at identifying PrRP analogues acting as either agonists or antagonists at the Hgr3 receptor. Such PrRP analogues could be useful receptorial tools able to clarify the multiple biological functions hypothesized for the PrRP receptor in the central nervous system.