G protein-coupled receptor kinase 6 (GRK6) plays a universal role in receptor desensitization, by acting as a receptor-G protein interface, thereby affecting serine/threonine kinases. In this study, a 20-aa-long peptide of human GRK6 C-terminus domain was synthesized and covalently coupled to keyhole limpet hemocyanin (KLH). A mouse monoclonal antibody against human GRK6 (anti-GRK6 MAb) was successfully prepared through hybridoma technique by immunizing BALB/c mice with synthesized GRK6426-446-KLH peptides. A high specificity and affinity strain of hybridoma 5D12 were established. The titer of the purified anti-GRK6 MAb was 1.28 × 10(6) measured by indirect ELISA. Western blot and immunocytochemistry experiments were also applied to characterize the antibody specificity. Antibody absorption assays showed that the anti-GRK6 MAb can be blocked by GRK6426-446 peptides. These results indicated that the antibody could bind to GRK6 antigen specifically. This MAb provides valuable support for further studies on the functional properties of GRK6.