Three major classes of opioid receptors, mu(mu), delta (delta) and kappa (kappa), have been defined based on differences in their pharmacology, physiology and tissue distribution. When stimulated in vivo the opioid receptors activate a cascade of intracellular reactions involving adenylyl cyclase, calcium channels and potassium channels, which result in many of the classical effects of opiate intoxication including euphoria, analgesia and physical dependence. The molecular characterization of the opioid receptors has been slow due to several factors, perhaps the most important of which being that they are intrinsic membrane proteins which are difficult to solubilize in active form and they are expressed in relatively low amounts. Recently these difficulties were overcome by the expression cloning of a mouse delta opioid receptor subtype from the neuroblastoma X glioma cell line NG108- 15. The primary known as the G protein-coupled receptors. In light of these recent reports we re-examined the sequence of an orphan receptor clone which we had obtained by degenerate PCR. This cDNA clone, referred to as R21, encodes a novel G protein-coupled receptor which shares significant sequence identity with the recently published mouse delta opioid receptor. Based on the conservation of key amino acid residues and the overall homology between R21 and the mouse delta opioid receptor we predict that R21 is a member of the opioid receptor family. To test this hypothesis we propose to pharmacologically characterize the receptor encoded by R21 and investigate the affect its stimulation has on adenylyl cyclase and a voltage-dependent outwardly rectifying potassium conductance. Once pharmacologically defined the tissue regulated the rat R21 gene will be characterized. The human homologue of the R21 gene will also be characterized to identify markers that can be used i genetic linkage and association studies of HR21 and human disease. Eventually the mouse gene, MR21, will be target and knocked out. These transgenic mice will be a valuable model system in which to evaluate the receptor's role in processes ranging from synaptic transmission to behavior.