Murine gammaherpesvirus 68 (MHV68) is a gammaherpesvirus that was first isolated from murid rodents. MHV68 establishes a latent infection in the spleen and other lymphoid organs. Several gammaherpesviruses, including herpesvirus saimiri, human herpesvirus 8, and MHV68, encode proteins with extensive homology to the D-type cyclins. To study the function of the cyclin homologue, a recombinant MHV68 has been constructed that lacks the cyclin homologue and expresses β-galactosidase as a marker (MHV68^cy−). MHV68^cy− grows in vitro with kinetics and to titers similar to those of the wild type. BALB/c mice infected with mixtures of equivalent amounts of the wild type and MHV68^cy− show deficient growth of the MHV68^cy− in an acute infection. Infection of SCID mice with virus mixtures also showed decreased MHV68^cy−virus growth, indicating that the deficiency is not mediated by T or B cells. Although mice infected with mixtures containing 100 times as much MHV68^cy− had greater splenic titers of the mutant virus than wild-type virus in acute infection, at 28 days postinfection splenocytes from these mice reactivated primarily wild-type virus. Quantitative PCR data indicate that equivalent genomes were present in the latent state. Reinsertion of the cyclin homologue into the cyclin-deleted virus restored the wild-type phenotype. These results indicate that the MHV68 cyclin D homologue mediates important functions in the acute infection and is required for efficient reactivation from latency.