Infection of mice with murine gammaherpesvirus 68 (MHV-68) robustly activates CD8 T cells, but only six class I major histocompatibility complex (MHC)-restricted epitopes have been described to date for the widely used H-2(b) haplotype mice. To explore the specificity and kinetics of the cytotoxic T-lymphocyte response in MHV-68-infected C57BL/6 mice, we screened for H-2K(b)- and H-2D(b)-restricted epitopes using a set of 384 candidate epitopes in an MHC tetramer-based approach and identified 19 new epitopes in 16 different open reading frames. Of the six known H-2K(b)- and H-2D(b)-restricted epitopes, we confirmed a response against three and did not detect CD8 T-cell-specific responses for the remaining three. The peak of the CD8 T-cell response to most peptides occurs between 6 and 10 days postinfection. The respective MHC tetramer-positive CD8 T cells display an activated/effector phenotype (CD62L(lo) and CD44(hi)) and produce gamma interferon upon peptide stimulation ex vivo. MHV-68 infection in vivo elicits a response to multiple viral epitopes, derived from both early and late viral antigens, illustrating a far broader T-cell repertoire and more-rapid activation than those previously recorded.