Abstract The effects of repeated s.c. administrations of an μ-opioid receptor antagonist naloxone on the G-protein activation induced by μ-opioid receptor agonists [ d-Ala 2, N-MePhe 4,Gly-ol 5]enkephalin (DAMGO), endomorphin-1 and endomorphin-2 in the mouse spinal cord was studied, monitoring guanosine-5′- o-(3-[ 35S]thio)triphosphate ([ 35S]GTPγS) binding. All μ-opioid receptor agonists concentration-dependently increased the [ 35S]GTPγS binding. The increases of [ 35S]GTPγS binding induced by agonists were significantly enhanced in mice pretreated with naloxone. Under the present condition, chronic treatment with naloxone significantly increased the density of [ 3H]DAMGO binding sites with an increase in K d values in spinal cord membranes, indicating an increase in μ-opioid receptors on the membrane surface. These findings suggest that chronic treatment with an μ-opioid receptor antagonist naloxone leads to the supersensitivity to activate G-protein by μ-opioid receptor agonists with an increase in μ-opioid receptors in membranes of the mouse spinal cord.