The neuropeptides orexin A and B (also known as hypocretins) play an important role in many physiological and behavioral activities. Orexins are ligands of two closely related G-protein-coupled receptors, that are the named orexin 1 and orexin 2 receptors. To clearly identify the minimal ligand sequences required for receptor activation, we synthesized and analyzed different centrally, C- and N-terminally truncated analogues of orexins A and B. Furthermore, we used the shortest active analogue to screen for important amino acid residues by l-alanine and l-proline replacement scans. For orexin A, only full-length peptides were able to show the same activity as orexin A, but interestingly, reduced orexin A and natural orexin A, which contains the two disulfide bonds, had the same activity. The shortest highly active orexin B analogue was orexin B 6-28. In addition, we identified orexin A 2-33 as the first analogue with orexin 1 receptor preference and orexin B 10-28, [A27]orexin B 6-28, and [P11]orexin B 6-28 as being highly potent orexin 2 receptor selective (>1000-fold) peptides.