Although the role of CD28-B7 interaction in the activation of naive T cells is well established, its importance in the generation and maintenance of T cell memory is not well understood. In this study, we examined the requirement for CD28-B7 interactions in primary T cell activation and immune memory. Ag-specific CD8 T cell responses were compared between wild-type (+/+) and CD28-deficient (CD28−/−) mice following an acute infection with lymphocytic choriomeningitis virus (LCMV). During the primary response, there was a substantial activation and expansion of LCMV-specific CD8 T cells in both+/+ and CD28−/− mice. However, the magnitude of the primary CD8 T cell response to both dominant and subdominant LCMV CTL epitopes was∼ 2-to 3-fold lower in CD28−/− mice compared with+/+ mice; the lack of CD28-mediated costimulation did not lead to preferential suppression of CD8 T cell responses to the weaker subdominant epitopes. As seen in CD28−/− mice, blockade of B7-mediated costimulation by CTLA4-Ig treatment of+/+ mice also resulted in a 2-fold reduction in the anti-LCMV CD8 T cell responses. Loss of CD28/B7 interactions did not significantly affect the generation and maintenance of CD8 T cell memory; the magnitude of CD8 T cell memory was∼ 2-fold lower in CD28−/− mice as compared with+/+ mice. Further, in CD28−/− mice, LCMV-specific memory CD8 T cells showed normal homeostatic proliferation in vivo and also conferred protective immunity. Therefore, CD28 signaling is not necessary for the proliferative renewal and maintenance of memory CD8 T cells.