The costimulatory molecules CD80 and CD86 (B7-1 and B7-2) are upregulated on mature antigen-presenting cells and interact with positive and negative regulators of CD8 T cell function, CD28 and CD152 (CTLA4) respectively. In this study, we examined the role of CD80 and CD86 in the immune response to murine gammaherpesvirus-68 (MHV-68) using CD80/86-/- mice. As we had previously shown that CD28 (the only known activating receptor for CD80 and 86) is not essential for long-term control of MHV-68, we predicted that CD80 and 86 would also be dispensable for an effective response to this virus. However, surprisingly, we observed that CD80/86-/- mice failed to maintain effective long-term control of MHV-68 and showed viral reactivation in the lungs. We did not observe viral reactivation in mice deficient in either CD80 or CD86 alone, indicating that these molecules play overlapping roles in the long-term control of MHV-68. Antiviral antibody responses were dramatically reduced in CD80/86-/- mice, while CD8 T cell expansion and recruitment to the lungs were not significantly affected. The unexpected disparity in the requirement for CD28 and CD80/86 in the response to MHV-68 suggests that CD28 is not the only positive regulatory receptor for CD80/86.