The cytotoxic T-lymphocyte response to wild-type simian virus 40 large tumor antigen (Tag) in C57BL/6 (H2^b ) mice is directed against three H2-D^b -restricted epitopes, I, II/III, and V, and oneH2-K^b -restricted epitope, IV. Epitopes I, II/III, and IV are immunodominant, while epitope V is immunorecessive. We investigated whether this hierarchical response was established in vivo or was due to differential expansion in vitro by using direct enumeration of CD8⁺ T lymphocytes with Tag epitope/major histocompatibility complex class I tetramers and intracellular gamma interferon staining. The results demonstrate that epitope IV-specific CD8⁺ T cells dominated the Tag-specific response in vivo following immunization with full-length Tag while CD8⁺ T cells specific for epitopes I and II/III were detected at less than one-third of this level. The immunorecessive nature of epitope V was apparent in vivo, since epitope V-specific CD8⁺ T cells were undetectable following immunization with full-length Tag. In contrast, high levels of epitope V-specific CD8⁺ T lymphocytes were recruited in vivo following immunization and boosting with a Tag variant in which epitopes I, II/III, and IV had been inactivated. In addition, analysis of the T-cell receptor β (TCRβ) repertoire of Tag epitope-specific CD8⁺ cells revealed that multiple TCRβ variable regions were utilized for each epitope except Tag epitope II/III, which was limited to TCRβ10 usage. These results indicate that the hierarchy of Tag epitope-specific CD8⁺T-cell responses is established in vivo.