A cDNA was isolated from rat brain by low stringency hybridization with the rat mu opioid receptor cDNA. Sequence analysis of this clone indicated that it contains an open reading frame capable of encoding a 367 amino acid protein. The deduced amino acid sequence of this protein shows high degrees of homology to all three opioid receptors, mu, kappa, and delta, suggesting that it is a member of the opioid receptor gene family. RNA blot analysis detected high level expression of the receptor mRNA in the brain. Southern blot analysis suggests that it is a single-copy gene, and mapping studies localized the gene on mouse chromosome 2. Despite the high sequence homologies between this protein and the other opioid receptors, expression studies of this clone in COS-7 cells did not show binding to [3H]diprenorphine, a ligand that binds to the other three opioid receptors. Furthermore, co-expression of this receptor with a G protein-activated potassium channel in Xenopus oocytes did not show functional coupling upon stimulation with mu, kappa and delta agonists. Given the similar degrees of high homology to the mu, kappa and delta opioid receptors and the lack of apparent affinity for their ligands, this receptor does not appear to belong to any of the three known classes of opioid receptors. Rather, it represents a novel member of the opioid receptor gene family, not identified from previous pharmacological studies.