Tyr-W-MIF-1 (Tyr-Pro-Trp-Gly-NH2) is a naturally occurring neuropeptide that displays high selectivity for mu-opioid receptors. Recently, intrathecal (i.t.) Tyr-W-MIF-1 was shown to induce potent analgesia mediated through spinal mu2-opioid receptors in mice. In the current study, we investigated the supraspinal analgesic effects of Tyr-W-MIF-1 using intracerebroventricular (i.c.v.) administration in mice. I.c.v. Tyr-W-MIF-1 induced a dose-dependent analgesic response with an ED50 of 31.4 micrograms that was antagonized by i.c.v. naloxone (ED50 = 4.46 nmol) and the mu-opioid receptor antagonist beta-funaltrexamine but not by the mu1-opioid receptor-selective antagonist naloxonazine. I.t. naloxone (ED50 = 0.12 nmol), however, was nearly 40-fold more potent than i.c.v. naloxone at antagonizing i.c.v. Tyr-W-MIF-1-induced analgesia. Tyr-W-MIF-1 also possesses antagonist activity at mu1-opioid receptors in brain. Coadministration of i.c.v. Tyr-W-MIF-1 with i.c.v. morphine or i.c.v. [D-Ala2, MePhe4, Gly(ol)5]enkephalin (DAMGO) significantly decreased the analgesic response to either drug administered alone. Thus, Tyr-W-MIF-1 functions as a mixed mu2-opioid receptor agonist/mu1-opioid receptor antagonist after i.c.v. administration in mice.