To help characterize K~ receptors and establish their relationship t o traditional p and 6 receptors, we have generated a K3-selective monoclonal antibody. Monoclonal antibodies were raised against BE(2)-C cells, a human neuroblastoma cell line containing p, K ~, and 6 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 ( K ~) binding without affecting 3H-DAMG0 ( p ) or 3H-naltrindole (6) 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 K3 agonist NalBzoH but not the p agonist morphine. Monoclonal antibody 8D8 (mAb8D8) also recognizes K~ receptors from mouse, rat, and calf brain. Administered intracerebroventricularly, mAb8D8 blocked K~ but not morphine (p) 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 K~ receptors and provide additional support for the existence of this unique opioid receptor subtype.