Background and Purpose: The macrocyclic tetrapeptide natural product CJ-15,208 (cyclo[Phe-D-Pro-Phe-Trp]) is a multifunctional μ-opioid receptor and κ-opioid receptor agonist and κ-opioid receptor antagonist that produces antinociception and prevents stress-induced reinstatement of extinguished cocaine-conditioned place preference (CPP). We hypothesized that an analogue of CJ-15,208, cyclo[Pro-Sar-Phe-D-Phe], would demonstrate multifunctional μ-opioid receptor and κ-opioid receptor ligand activity, producing potent antinociception with fewer liabilities than selective μ-opioid receptor agonists, while preventing both drug-and stress-induced reinstatement of morphine-induced CPP. Experimental Approach: The opioid receptor agonist and antagonist activity of cyclo [Pro-Sar-Phe-D-Phe] was characterized after i.c.v. and i.p. administration to C57BL/6J or transgenic opioid receptor "knockout" mice using the 55 C warm-water tail-withdrawal assay. Liabilities of locomotor coordination, respiration and spontaneous ambulation, and direct rewarding or aversive properties were assessed. Finally, the ability of cyclo[Pro-Sar-Phe-D-Phe] to block morphine-and stress-induced reinstatement of extinguished CPP was determined. Key Results: cyclo[Pro-Sar-Phe-D-Phe] demonstrated dose-dependent, short-lasting antinociception, with an ED 50 (and 95% confidence interval) of 0.15 (0.05-0.21) nmol i.c.v. and 1.91 (0.40-3.54) mgÁkg −1 i.p., mediated by μand κ-opioid receptors. The macrocyclic tetrapeptide also demonstrated potent dose-dependent κ-opioid receptor antagonist-like activity at 2.5, but not at 4.5, h after administration. cyclo[Pro-Sar-Phe-D-Phe] displayed reduced liabiities compared with morphine, attributed to its additional activity at κ-receptors. Pretreatment with cyclo[Pro-Sar-Phe-D-Phe] prevented stress-and drug-induced reinstatement of extinguished morphine-place preference responses in a time-dependent manner.