In the present study we investigated and compared the in vivo analgesia of centrally administered endomorphin-2 and morphiceptin, and their analogs modified in position 3. Two series of analogs were synthesized by introducing unnatural aromatic amino acids in the D configuration: 3-(1-naphthyl)-D-alanine (D-1-Nal), 3-(2-naphthyl)-D-alanine (D-2-Nal), 3-(4-chlorophenyl)-D-alanine (D-ClPhe), 3-(3,4-dichlorophenyl)-D-alanine (D-Cl2Phe). Antinociceptive activity of endomorphin-2, morphiceptin, and their analogs was compared in the mouse hot-plate test, performed after i.c.v. administration of the peptides at a dose of 10 microg/animal. The best results were obtained for two morphiceptin analogs, [D-Phe3]morphiceptin and [D-1-Nal3]morphiceptin, which showed greatly improved analgesic activity, as compared to morphiceptin. In the endomorphin-2 series none of the modifications produced analogs more potent than the parent compound, but [D-1-Nal3]endomorphin-2 was the best analog. Antinociception induced by endomorphin-2 was reversed by concomitant i.c.v. administration of [D-Phe3]endomorphin-2, [D-2-Nal3]endomorphin-2, and [D-2-Nal3]morphiceptin, indicating that these analogs were weak mu-opioid antagonists.