Abstract The spike (S) protein of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is not only responsible for receptor binding, but also a major antigenic determinant capable of inducing protective immunity. In this study, we demonstrated that the receptor-binding domain (RBD) of S protein is an important immunogenic site in patients with SARS and rabbits immunized with inactivated SARS-CoV. Serum samples from convalescent SARS patients and immunized rabbits had potent neutralizing activities against infection by pseudovirus expressing SARS-CoV S protein. Depletion of RBD-specific antibodies from patient or rabbit immune sera by immunoadsorption significantly reduced serum-mediated neutralizing activity, while affinity-purified anti-RBD antibodies had relatively higher potency neutralizing infectivity of SARS pseudovirus, indicating that the RBD of S protein is a critical neutralization determinant of SARS-CoV during viral infection and immunization. Two monoclonal antibodies (1A5 and 2C5) targeting at the RBD of S protein were isolated from mice immunized with inactivated SARS-CoV. Both 1A5 and 2C5 possessed potent neutralizing activities, although they directed against distinct conformation-dependant epitopes as shown by ELISA and binding competition assay. We further demonstrated that 2C5, but not 1A5, was able to block binding of the RBD to angiotensin-converting enzyme 2 (ACE2), the functional receptor on targeted cells. These data provide important information for understanding the antigenicity and immunogenicity of SARS-CoV and for designing SARS vaccines.