Recent studies have demonstrated that CD28 provides critical costimulatory signals required for optimal CD8 T cell expansion and effector function in response to several viruses, including influenza, HSV, and vaccinia virus (VACV). CD28 has two ligands expressed largely on professional APC, named B7.1 (CD80) and B7.2 (CD86). Although some results suggest that these ligands are equivalent and both promote CD28 signaling, it is not clear whether they are equally important for priming of antiviral T cells. Herein we show that B7.2 is critical for early CD8 T cell responses to both dominant and subdominant VACV epitopes, correlating with its strong induction on CD8alpha(+) dendritic cells. In contrast, B7.1 plays no significant role. Signals from an exogenously applied adjuvant can recruit B7.1 activity and lead to further enhanced priming of VACV-reactive CD8 T cells. However, during a natural infection, B7.1 is not functional, likely related to inefficient up-regulation or active suppression by VACV. These studies provide evidence that B7.2 is the major ligand for the CD28 receptor on VACV-specific CD8 T cells, that B7.2 can promote efficient CD8 T cell priming without B7.1, and that B7.1 and B7.2 can be differentially utilized during antiviral responses.