Infection of mice with the intracellular bacterium Listeria monocytogenes results in a strong CD8+ T cell response that is critical for efficient control of infection. We used CD28-deficient mice to characterize the function of CD28 during Listeria infection, with a main emphasis on Listeria-specific CD8+ T cells. Frequencies and effector functions of these T cells were determined using MHC class I tetramers, single cell IFN-γ production and Listeria-specific cytotoxicity. During primary Listeria infection of CD28−/− mice we observed significantly reduced numbers of Listeria-specific CD8+ T cells and only marginal levels of specific IFN-γ production and cytotoxicity. Although frequencies were also reduced in CD28−/− mice during secondary response, we detected a considerable population of Listeria-specific CD8+ T cells in these mice. In parallel, IFN-γ production and cytotoxicity were observed, revealing that Listeria-specific CD8+ T cells in CD28−/− mice expressed normal effector functions. Consistent with their impaired CD8+ T cell activation, CD28−/− mice suffered from exacerbated listeriosis both after primary and secondary infection. These results demonstrate participation of CD28 signaling in the generation and expansion of Ag-specific CD8+ T cells in listeriosis. However, Ag-specific CD8+ T cells generated in the absence of CD28 differentiated into normal effector and memory T cells.