The present study was designed to investigate the possible change in spinal µ-opioid receptor function after repeated administration of a selective κ-opioid receptor agonist (1S-trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl) cyclohexyl]-benzeneacetamide hydrochloride [(−)U-50,488H] in the ICR mouse. A single s.c. or i.t. injection of (−)U-50,488H produced a dose-dependent antinociception. Repeated s.c. or i.t. administration of (−)U-50,488H resulted in the development of tolerance to (−)U-50,488H-induced antinociception. Under these conditions, we demonstrated here that repeated s.c. injection of (−)U-50,488H significantly enhanced the antinociceptive effect induced by the i.t. administration of a selective µ-opioid receptor agonist [d-Ala2,N-Me-Phe4,Gly5-ol] enkephalin (DAMGO). Using the guanosine-5′-o-(3-[35S]thio) triphosphate ([35S]GTPγS) binding assay, we found that (−)U-50,488H was able to produce a dose-dependent increase in [35S]GTPγS binding to membranes of the mouse spinal cord. Repeated administration of (−)U-50,488H caused a significant reduction in the (−)U-50,488H-stimulated [35S]GTPγS binding in this region, whereas repeated treatment with (−)U-50,488H exhibited an increase in the DAMGO-stimulated [35S]GTPγS binding in membranes of the spinal cord. Using a receptor binding assay, repeated treatment with (−)U-50,488H significantly increased the density of [3H]DAMGO binding sites in membranes of the mouse spinal cord. In contrast, the expression of µ-opioid receptor was not affected after repeated treatment with (−)U-50,488H. These results suggest that repeated stimulation of κ-opioid receptors leads to the up-regulation of µ-opioid receptor functions in the spinal cord, which may be associated with an increase in the number of functional µ-opioid receptors in the mouse spinal cord.