Vertebrates express three cytokine-inducible proteasome subunits that are incorporated in the place of their constitutively synthesized counterparts. There is increasing evidence that the set of peptides generated by proteasomes containing these subunits (immunoproteasomes) differs from that produced by standard proteasomes. In this study, we use mice lacking one of the immunoproteasome subunits (LMP2) to show that immunoproteasomes play an important role in establishing the immunodominance hierarchy of CD8(+) T cells (T(CD8+)) responding to seven defined determinants in influenza virus. In LMP2(-/)- mice, responses to the two most dominant determinants drop precipitously, whereas responses to two subdominant determinants are greatly enhanced. Adoptive transfer experiments with naive normal and transgenic T(CD8+) reveal that the reduced immunogenicity of one determinant (PA(224-233)) can be attributed to decreased generation by antigen presenting cells (APCs), whereas the other determinant (NP(366-374)) is less immunogenic due to alterations in the T(CD8+) repertoire, and not, as reported previously, to the decreased capacity of LMP2(-/)- APCs to generate the determinant. The enhanced response to one of the subdominant determinants (PB1F2(62-70)) correlates with increased generation by LMP2(-)(/)- virus-infected cells. These findings indicate that in addition to their effects on the presentation of foreign antigens, immunoproteasomes influence T(CD8+) responses by modifying the repertoire of responding T(CD8+).