Cyclic analogs of the opioid peptides endomorphin-2 and morphiceptin of the type Tyr-X-Phe-Phe-Y-NH2 and Tyr-X-Phe-D-Pro-Y-NH2 (X = Lys or Asp and Y = Lys or Asp), respectively, were synthesized in order to test their structure-activity relationships. Antinociceptive activity of the new analogs was assessed in the hot-plate test after intracerebroventricular administration in mice. The strong analgesic effect was observed for the analogs with Asp in position 2, while the analogs with Lys in the second position were inactive. Antinociception caused by Asp2 analogs was dose-dependent and reversed by the concomitant administration of the universal opioid antagonist naloxone and by the selective kappa antagonist, nor-BNI. However, receptor binding studies revealed poor affinity of all cyclic analogs at the mu-opioid receptor and no affinity at delta- and kappa-opioid receptors. It is most likely that the new cyclic analogs produced their antinociception by the release of dynorphin A, which subsequently acted on the kappa-opioid receptor.