Open reading frame 24 (ORF24) of murine gammaherpesvirus 68 (MHV-68) is conserved among beta- and gammaherpesviruses; however, its function in viral replication has not been defined. Using MHV-68 as a model, we have identified ORF24 as being essential for viral replication. An ORF24-null virus was generated and shown to be defective in late gene expression. Expression of early genes, as well as viral genome replication, was not affected. Furthermore, the defect in late gene expression was likely due to a deficiency in transcription. Thus, we have identified an MHV-68 protein, ORF24, that is essential for the expression of viral late proteins yet dispensable for viral DNA replication.