The ability of CD8(+) T cells to engage a diverse range of peptide-major histocompatibility complex (MHC) complexes can also lead to cross-recognition of self and nonself peptide-MHC complexes and thus directly contribute toward allograft rejection or autoimmunity. Here we present a novel form of cross-recognition by herpes virus-specific CD8(+) cytotoxic T cells that challenges the current paradigm of self/non-self recognition. Functional characterization of a human leukocyte antigen (HLA) Cw*0602-restricted cytomegalovirus-specific CD8(+) T-cell response revealed an unusual dual specificity toward a pp65 epitope and the alloantigen HLA DR4. This cross-recognition of HLA DR4 alloantigen was critically dependent on the coexpression of HLA DM and was preferentially directed toward the B-cell lineage. Furthermore, allostimulation of peripheral blood lymphocytes with HLA DRB*0401-expressing cells rapidly expanded CD8(+) T cells, which recognized the pp65 epitope in the context of HLA Cw*0602. T-cell repertoire analysis revealed 2 dominant populations expressing T-cell receptor beta variable (TRBV)4-3 or TRBV13, with cross-reactivity exclusively mediated by the TRBV13(+) clonotypes. More importantly, cross-reactive TRBV13(+) clonotypes displayed markedly lower T-cell receptor binding affinity and a distinct pattern of peptide recognition, presumably mimicking a structure presented on the HLA DR4 allotype. These results illustrate a novel mechanism whereby virus-specific CD8(+) T cells can cross-recognize HLA class II molecules and may contribute toward allograft rejection and/or autoimmunity.