To investigate the functional role of G protein-coupled receptor kinases (GRK) in homologous desensitization of the mu-opioid receptor, human embryonic kidney (HEK) 293 cells, which express a significant level of GRK2, were stably transfected with the cDNA encoding the rat mu-opioid receptor. Wild-type mu-opioid receptors developed homologous desensitization after 30 min pretreatment with DAMGO ([D-Ala2,N-methyl-Phe4,Gly-ol5]enkephalin), a specific mu-opioid receptor agonist. The ability of mu-opioid receptors to develop homologous desensitization was greatly impaired following the transfection of a cDNA fragment encoding the GRK2(495-689) polypeptide, which is believed to block Gbetagamma-mediated transduction events including the membrane translocation and activation of GRK2. The mu(Cdelta45) receptor, a deletion mutant that lacks 45 C-terminal amino acids, failed to exhibit homologous desensitization after 30 min pretreatment of DAMGO. The mu(Cdelta41) receptor, which differs from the mu(Cdelta45) receptor by having four more Ser/Thr residues (Thr354Ser355Ser356Thr357), developed GRK2-mediated desensitization. These results suggest that homologous desensitization of rat mu-opioid receptors results from the activation of GRK2 and that a cluster of Ser/Thr residues (Thr354Ser355Ser356Thr357) at the intracellular carboxyl tail plays an important role in GRK2-mediated mu-opioid receptor desensitization.