Receptor binding properties and antinociceptive activities of chimeric peptides linked by spacers were investigated. The peptides consisted of the μ-opioid receptor ligand dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH₂) or its analog YRFB (Tyr-D-Arg-Phe-βAla-NH₂) linked to the ORL1 receptor ligand Ac-Arg-Tyr-Tyr-Arg-Ile-Lys-NH₂ (Ac-RYYRIK-NH₂). All chimeric peptides were found to possess high receptor binding affinities for both μ-opioid and ORL1 receptors in mouse brain membranes although their binding affinities for both receptors in spinal membranes were significantly lower. Among them, chimeric peptide 2, which consists of dermorphin and Ac-RYYRIK-NH₂ connected by a long spacer, had the highest binding affinity towards both receptors. In the tail-flick test following intrathecal (i.t.) administration to mice, all chimeric peptides showed potent and dose-dependent antinociceptive activities with an ED₅₀ of 1.34—4.51 (pmol/mouse), nearly comparable to dermorphin alone (ED₅₀; 1.08 pmol/mouse). In contrast to their μ-opioid receptor binding profiles, intracerebroventricular (i.c.v.) administration of the chimeric peptides resulted in much less potent antinociceptive activity (ED₅₀ 5.55—100< pmol/mouse) than when administered i.t. (ED₅₀: 1.34—4.51 pmol/mouse). These results suggest the involvement of nociceptin-like agonistic effects of the Ac-RYYRIK pharmacophore in the peptides, and the regulation of μ-opioid receptor-mediated antinociception in brain. The present chimeric peptides may be useful as pharmacological tools for studies on μ-opioid receptor/ORL1 receptor heterodimers.