The present study was designed to investigate the possible change in spinal mu-opioid receptor function after repeated administration of a selective kappa-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 mu-opioid receptor agonist [D-Ala(2),N-Me-Phe(4),Gly(5)-ol] enkephalin (DAMGO). Using the guanosine-5'-o-(3-[S-35]thio) triphosphate ([S-35]GTPgammaS) binding assay, we found that (-)U-50,488H was able to produce a dose-dependent increase in [S-35]GTPgammaS binding to membranes of the mouse spinal cord. Repeated administration of (-)U-50,488H caused a significant reduction in the (-)U-50,488H-stimulated [S-35]GTPgammaS binding in this region, whereas repeated treatment with (-)U-50,488H exhibited an increase in the DAMGO-stimulated [S-35]GTPgammaS binding in membranes of the spinal cord. Using a receptor binding assay, repeated treatment with (-)U-50,488H significantly increased the density of [H-3]DAMGO binding sites in membranes of the mouse spinal cord. In contrast, the expression of mu-opioid receptor was not affected after repeated treatment with (-)U-50,488H. These results suggest that repeated stimulation of kappa-opioid receptors leads to the up-regulation of mu-opioid receptor functions in the spinal cord, which may be associated with an increase in the number of functional mu-opioid receptors in the mouse spinal cord.