Rhesus monkey rhadinovirus (RRV) is a gammaherpesvirus that is closely related to human Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8). RRV is the closest relative to KSHV that has a fully sequenced genome and serves as an in vitro and an in vivo model system for KSHV. The latency-associated nuclear antigen (LANA) protein of both KSHV and RRV plays key roles in the establishment and maintenance of these herpesviruses. We have constructed a RRV recombinant virus (RRVDeltaLANA/GFP) in which the RRV LANA open reading frame has been disrupted with a green fluorescent protein (GFP) expression cassette generated by homologous recombination. The integrity of the recombinant virus was confirmed by diagnostic PCR, restriction digestion, Southern blot analysis, and whole-genome sequencing. We compared the single-step and multistep replication kinetics of RRVDeltaLANA/GFP, RRV-GFP, wild-type (WT) RRV H26-95, and a revertant virus using traditional plaque assays, as well as real-time quantitative PCR-based genome quantification assays. The RRVDeltaLANA/GFP recombinant virus exhibited significantly higher lytic replicative properties compared to RRV-GFP, WT RRV, or the revertant virus. This was observed upon de novo infection and in the absence of chemical inducers such as phorbol esters. In addition, by using a quantitative real-time PCR-based viral array, we are the first to report differences in global viral gene expression between WT and recombinant viruses. The RRVDeltaLANA/GFP virus displayed increased lytic gene transcription at all time points postinfection compared to RRV-GFP. Moreover, we also examined several cellular genes that are known to be repressed by KSHV LANA and report that these genes are derepressed during de novo lytic infection with the RRVDeltaLANA/GFP virus compared to RRV-GFP. Finally, we also demonstrate that the RRVDeltaLANA/GFP virus fails to establish latency in B cells, as measured by the loss of GFP-positive cells and intracellular viral genomes.