The spinal cord contains μ, δ and κ opioid receptors which mediate the antinociceptive effects of opioid agonists administered onto the spinal cord. In this study, we characterized the binding sites for highly-selective μ, δ and κ opioid radioligands and quantified and distribution of opioid binding sites in rat lumbosacral spinal cord using autoradiography. In sections of rat brain mounted on glass slides, the μ ligand, [ 3H]sufentanil, bound with high affinity with an apparentK d of 0.46 nM. The δ ligand, [ 3H]DPDPE ([ d-Pen 2,5]-enkephalin), bound with aK d of 4.31 nM, and the κ-ligand, [ 3H]U69593, bound with aK d of 2.27 nM. Three regions of the spinal gray were targeted for quantification of binding sites by autoradiography. The data indicate that when considered as a percentage of the total opioid binding capacity within a region, the contribution of μ sites in laminae I-II was about 90%, with δ and κ sites 7% and 3%, respectively. In lamina V, the μ sites comprised about 70% of the total opioid sites, with δ and κ sites comprising 28% and 2%, respectively. In the area adjacent to the central canal, μ sites contributed about 65% of the total opioid sites followed by δ sites at 33% and κ sites at 2% of total opioid sites. These results demonstrate a differential distribution of μ, δ and κ binding sites with respect to the organization of the spinal gray matter. The preferential occurrence of all 3 opioid binding sites in the superficial dorsal horn is noteworthy since many fine caliber primary afferent fibers mediating nociception establish synaptic contact in this region.