Previous studies from our laboratory have shown that systemic chronic morphine treatment causes down-regulation of spinal adenosine A1 receptors in rats. In this study, we further investigated whether supraspinal morphine treatment causes this effect. Adult male Sprague-Dawley rats were rendered tolerant to morphine by multiple intracerebroventricular (i.c.v.) injections for 2 or 4 days. Adenosine A1 receptor binding activities were measured with [3H]cyclohexyladenosine in the spinal cord and midbrain. A significant decrease in [3H]cyclohexyladenosine binding was found in the spinal cord but not in the midbrain region after 2 or 4 days of chronic i.c.v. morphine treatment. A decrease in the number of binding sites (Bmax) with no change in the affinity (Kd) of the ligand for the adenosine A1 receptor was observed. These results suggest that supraspinal morphine administration could cause the down-regulation of spinal adenosine A1 receptors and this may play a role in the mechanism of morphine tolerance.