Severe acute respiratory syndrome (SARS) is a newly emerged human infectious disease caused by the severe acute respiratory syndrome coronavirus (SARS-CoV). The spike (S) protein of SARS-CoV is a major virion structural protein. It plays an important role in the interaction with receptors and neutralizing antibodies. Previously study demonstrated that amino acids 318 to 510 is the receptor binding domain of SARS-CoV spike protein. The receptor-binding domain of the spike protein was expressed by fusion with GST in a pGEX-6p-1 vector. Western blot results demonstrated that this fragment could be recognized by SARS convalescent sera and spike protein specific monoclonal antibody. To map the antigenic epitope of this region, a set of 23 partially overlapping fragments spanning the fragment were fused with GST and expressed. Then Western blot and ELISA reactivity of these short peptide fused protein to immunized sera and monoclonal antibody D3D1 were surveyed. Two linear antigenic epitopes SRBD3 (334~349) and D3D1 (447~455) were identified. Identification of antigenic epitopes of the spike protein of SARS-CoV may provide the basis for the development of immunity-based prophylactic, therapeutic, and diagnostic clinical techniques for severe acute respiratory syndrome.