kappa(3) opioid receptors have a unique binding and analgesic profile, as originally defined by naloxone benzoylhydrazone (NalBzoH). Although antisense studies demonstrated the close relationship between kappa(3) opioid and Orphan opioid receptor-like receptor (ORL1) and implied they were generated from the same gene, these studies also revealed differences in the sensitivity profiles of NalBzoH and orphanin FQ/nociceptin (OFQ/N), indicating that they were not identical. To help define the relationship between kappa(3) and ORL1 receptors, we utilized BE(2)-C human neuroblastoma cells that natively express functional ORL1 and kappa(3) opioid receptors. (125)I-[Tyr(14)]OFQ/N binds to a single population of receptors in BE(2)-C cells. Competition binding and adenylyl cyclase studies clearly illustrated marked selectivity differences between the ORL1 and the kappa(3) sites. Furthermore, antisense DNA targeting ORL1 blocked the inhibition of cAMP by OFQ/N, but not by NalBzoH. Thus, the receptor mechanisms mediating the activity of OFQ/N and NalBzoH in BE(2)-C cells are distinct.