Our functional mapping study of murine gammaherpesvirus 68 (MHV-68, or gammaHV-68) revealed that a mutant harboring a transposon at the ORF49 locus (ORF49(null)) evidenced a highly attenuated in vitro growth. ORF49 resides adjacent to and in an opposite direction from RTA, the primary switch of the gammaherpesvirus life cycle. A FLAG-tagged ORF49 protein was able to transcomplement ORF49(null), and a revertant of ORF49(null) restored its attenuated growth to a level comparable to that of the wild type. The FLAG-tagged ORF49 protein promoted the ability of RTA to activate downstream target promoters and enhanced virus replication from the ORF50(null) virus in the presence of RTA. Furthermore, ORF49 enhanced wild-type virus replication by increasing the RTA transcript levels. Our data indicate that ORF49 may perform an important function in MHV-68 replication in cooperation with RTA.