The causative agent of severe acute respiratory syndrome (SARS) has been identified as a new type of coronavirus, SARS-associated coronavirus (SARS-CoV). CD8 T cells play an important role in controlling diseases caused by other coronaviruses and in mediating vaccine-induced protective immunity in corresponding animal models. The spike protein, a main surface antigen of SARS-CoV, is one of the most important antigen candidates for vaccine design. Overlapping peptides were used to identify major histocompatibility complex class I-restricted epitopes in mice immunized with vectors encoding codon-optimized SARS-CoV spike protein. CD8 T-cell responses were mapped to two H-2(b)-restricted epitopes (S436-443 and S525-532) and one H-2(d)-restricted epitope (S366-374). The identification of these epitopes will facilitate the evaluation of vaccine strategies in murine models of SARS-CoV infection. Furthermore, codon and promoter optimizations can greatly enhance the overall immunogenicity of spike protein in the context of replication-defective human and simian adenoviral vaccine carriers. The optimized recombinant adenoviral vaccine vectors encoding spike can generate robust antigen-specific cellular immunity in mice and may potentially be useful for control of SARS-CoV infection.