To help characterize kappa3 receptors and establish their relationship to traditional mu and delta receptors, we have generated a kappa3-selective monoclonal antibody. Monoclonal antibodies were raised against BE(2)-C cells, a human neuroblastoma cell line containing mu, kappa3, and delta opioid receptors. Of the 5,000 hybridoma cell lines screened, approximately 2,000 hybridomas tested positive against BE(2)-C membranes by ELISA, but only 98 of these were negative against a different neuroblastoma cell line lacking opioid receptors. Supernatants from one hybridoma, 8D8, inhibited up to 90% of 3H-NalBzoH (kappa3) binding without affecting 3H-DAMGO (mu) or 3H-naltrindole (delta) binding in BE(2)-C membranes. The selectivity of the antibody was further demonstrated by its blockade of the inhibition of cAMP accumulation in BE(2)-C cells by the kappa3 agonist NalBzoH but not the mu agonist morphine. Monoclonal antibody 8D8 (mAb8D8) also recognizes kappa3 receptors from mouse, rat, and calf brain. Administered intracerebroventricularly, mAb8D8 blocked kappa3 but not morphine (mu) analgesia in vivo. On Western blots, mAb8D8 recognized a protein with a molecular mass of approximately 70 kilodaltons in BE(2)-C. These studies demonstrate the selectivity of mAb8D8 for kappa3 receptors and provide additional support for the existence of this unique opioid receptor subtype.