Previously, we reported on an opioid antagonist, nor-binaltorphimine (nor-BNI), that had high selectivity for kappa opioid receptors in smooth muscle preparations. In this study, nor-BNI administered either s.c. or i.c.v. was shown to antagonize significantly the antinociceptive effects of the kappa opioid agonists, ethylketazocine and U-50,488H at doses that had no effect on the antinociceptive effect of mu agonists, morphine and [D-Ala3, MePhe4, Gly-ol5]enkephalin and the delta agonist, [D-Pen3, D-Pen5]enkephalin. Nor-BNI and U-50,488H were used to demonstrate that kappa opioid receptors in the spinal cord were more important than those located supraspinally for kappa-mediated analgesia. Nor-BNI also possessed high affinity and high selectivity for kappa opioid receptors in the receptor binding assay. However, the comparatively low selectivity of BNI in receptor binding studies did not correlate with the high pharmacologic selectivity for kappa receptors.