Transcriptional silencing mediated by DNA methylation is a critical component of epigenetic regulation during early embryonic development in animals. However, the requirement for DNA methylation during activation and differentiation of mature CD8+ T cells into effector and memory cells is not clear. Using cre-mediated deletion of DNA methyltransferase 1 (Dnmt1) at the time of CD8+ T cell activation, we investigated the obligation for maintaining patterns of DNA methylation during the generation of Ag-specific effector and memory CD8+ T cells in response to acute viral infection of mice with lymphocytic choriomeningitis virus. Dnmt1-/- CD8+ T cells failed to undergo the massive CD8+ T cell expansion characteristic of lymphocytic choriomeningitis virus infection, leading to >80% reductions in Ag-specific effector CD8+ T cells at the height of the response. Despite this, Dnmt1-/- CD8+ T cells efficiently controlled the viral infection. Interestingly, the number of Ag-specific Dnmt1-/- memory CD8+ T cells was moderately reduced compared with the reductions seen at day 8 postinfection. Our data suggest that ablation of Dnmt1 and subsequent DNA methylation affect the finite proliferative potential of Ag-specific CD8+ T cells with moderate effects on their differentiation to effector and memory CD8+ T cells.