The Epstein-Barr virus (EBV) BMRF1 gene product is an essential component of the viral DNA polymerase and is absolutely required for lytic virus replication. In addition to its polymerase accessory protein function, we recently demonstrated that BMRF1 is a transactivator, inducing expression of the essential oriLyt promoter, BHLF1. However, the regions of BMRF1 required for transactivation of BHLF1 are unknown. Here we demonstrate that the carboxy-terminal portion of the BMRF1 protein (amino acids 378404), although not required for DNA binding or polymerase processivity function, is required for transactivator function as well as nuclear localization. Site-directed mutagenesis of this region allowed us to separate the transactivator and nuclear localization motifs of BMRF1. The two DNA-binding domains of BMRF1 are also required for efficient transactivation of the BHLF1 promoter.