Efficient induction of T cell responses is normally assumed to require both TCR-mediated signaling and engagement of co-stimulatory molecules, in particular CD28. However, the importance of CD28 co-stimulation in induction and maintenance of antiviral T cell responses is not clearly established. For this reason antiviral CD4(+) and CD8(+) T cell responses in CD28-deficient mice were studied using two different viruses [vesicular stomatitis virus and lymphocytic choriomeningitis virus (LCMV)]. Intracellular cytokine staining and/or MHC-peptide tetramers were used to enumerate antigen-specific T cells. In addition, we used DNA constructs encoding viral epitopes to probe the importance of the epitope itself. Our results reveal that while the context of antigen presentation (live virus versus DNA construct) is a critical factor in determining the requirement for CD28 co-stimulation, epitope and virus dose play little if any role. Direct visualization of antigen-specific cells also confirms the notion that CD28 is more critical for the generation of antiviral T(h)1 cells than for T(c)1 cells generated in response to the same virus (LCMV). Most importantly, the present study reveals that CD28 generally is essential for the host to respond optimally over a broad set of conditions, and our results may imply that the relatively CD28 independent activation of LCMV-specific CD8(+) T cells may represent an extreme situation related to the non-cytolytic nature of this virus allowing the delivery of a uniquely strong and prolonged signal 1.