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 CD8α+ 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.