We examined glycosylation of FLAG-hKOR expressed in CHO cells and determined its functional significance. FLAG-hKOR was resolved as a broad and diffuse 55-kDa band and a less diffuse 45-kDa band by immunoblotting, indicating that the receptor is glycosylated. Endoglycosidase H cleaved the 45-kDa band to approximately 38 kDa but did not change the 55-kDa band, demonstrating that the 45-kDa band is N-glycosylated with high-mannose or hybrid-type glycan. Peptide-N-glycosidase F digestion of solubilized hKOR or incubation of cells with tunicamycin resulted in two species of 43 and 38 kDa, suggesting that the 43-kDa band is O-glycosylated. FLAG-hKOR was reduced to lower Mr bands by neuraminidase and O-glycosidase, indicating that the hKOR contains O-linked glycan. Mutation of Asn25 or Asn39 to Gln in the N-terminal domain reduced the Mr by approximately 5 kDa, indicating that both residues were glycosylated. The double mutant hKOR-N25/39Q was resolved as a 43-kDa (mature form) and a 38-kDa (intermediate form) band. When transiently expressed, hKOR-N25/39Q had a lower expression level than the wild type. In CHO cells stably expressing the hKOR-N25/39Q, pulse-chase experiments revealed that the turnover rate constants (ke) of the intermediate and mature forms were approximately 3 times those of the wild type. In addition, the maturation rate constant (ka) of the 43-kDa form of hKOR-N25/39Q was 6 times that of the mature form of the wild type. The hKOR-N25/39Q mutant showed increased agonist-induced receptor phosphorylation, desensitization, internalization, and downregulation, without changing ligand binding affinity or receptor-G protein coupling. Thus, N-glycosylation of the hKOR plays important roles in stability and trafficking along the biosynthesis pathway of the receptor protein as well as agonist-induced receptor regulation.