Gammaherpesvirus pathogenesis is dependent on the ability of these viruses to establish a lifelong latent infection and the ability to reactivate from latency. Immediate-early genes of theses viruses are thought to be critical regulators of lytic replication and reactivation from latency. The gene 50-encoded Rta is the only immediate-early gene product that appears to be conserved among all characterized gammaherpesviruses. Previous studies have demonstrated that, in Epstein-Barr virus (EBV), Kaposi's sarcoma-associated virus, and gammaherpesvirus 68 (gamma HV68, also referred to as murine gammaherpesvirus 68), ectopic expression of Rta in latently infected cell lines can lead to induction of the viral cycle. Recently, studies employing null mutants of EBV have provided a formal demonstration that both Rta and the BZLF1 gene product, Zta, the two EBV immediate-early gene products, are essential for EBV replication. Here we generate and characterize a gene 50-null mutant gamma HV68 and demonstrate that the gene 50 product Rta is essential for virus replication. Providing gamma HV68 Rta in trans was sufficient to restore replication of the gene 50-null virus. Notably, Rta expressed from the spliced form of the gene 50 transcript was sufficient to complement growth of the gene 50-null virus. In addition, we provide evidence that loss of Rta expression leads to a complete defect in viral DNA replication and a significant defect in late antigen expression. This work lays the foundation for characterizing the role of Rta in gamma HV68 chronic infection of mice.