A new series of 12 dermorphin tetrapeptides, W-Tyr-D-MetO-Phe-Xaa-Y (W = H, H2NC = (NH); Xaa = Gly, Sar, D-Ala; Y = OH, OCH3, NH2) were prepared by traditional methods in solution and tested for opioid activity. In binding studies based on displacement of mu, delta, and kappa opioid receptor selective radiolabels from guinea pig brain membranes, the new analogues showed a negligible affinity for the kappa binding site and a preference for mu- over delta-receptors with an evident dependence on N- and/or C-terminal modifications; H-Tyr-D-MetO-Phe-Gly-OCH3 was shown to be one of the most selective mu-receptor ligands reported to date. All these tetrapeptides display dose-related naloxone-reversible antinociceptive effects following intracerebroventricular (icv) or subcutaneous (sc) administrations in mice. In comparison to morphine, H-Tyr-D-MetO-Phe-Sar-NH2 and the guanidino derivative H2NC = (NH)-Tyr-D-MetO-Phe-Gly-NH2 showed lower affinity for mu, delta, and kappa sites but exceptionally stronger analgesia: respectively they are 560 and 1550 times as potent an analgesic as morphine. Among analogues tested after sc administration, H-Tyr-D-MetO-Phe-Sar-NH2 and H-Tyr-D-MetO-Phe-D-Ala-OH displayed the highest activities; they were respectively 22 and 30 times more potent than morphine on a molar basis. These results indicate that N- or C-terminal modifications and substitution at position 2 or 4 of dermorphin-(1-4) peptide do not only influence the affinity of the resulting analogues to opioid receptors but also may favorably alter their pharmacokinetic properties.