The EBV DNA polymerase accessory protein, BMRF1, is an essential component of the viral DNA polymerase and is required for lytic EBV replication. In addition to its polymerase accessory protein function, we have recently reported that BMRF1 is a transcriptional activator, inducing expression of the essential oriLyt promoter, BHLF1. Here we have precisely mapped the BMRF1-response element in the BHLF1 promoter. We demonstrate that a region of oriLyt (the "downstream component"), previously shown to be one of two domains absolutely essential for oriLyt replication, is required for BMRF1-induced activation of the BHLF1 promoter. Furthermore, the downstream component of oriLyt is sufficient to confer BMRF1-responsiveness to a heterologous promoter. The downstream component contains Sp1 binding sites, and confers Sp1-responsiveness to a heterologous promoter. A series of plasmids containing various protions of the oriLyt downstream component were constructed and analyzed for their ability to respond to the BMRF1 versus Sp1 transactivators. Although the BMRF1-responsive region of the downstream component overlaps the Sp1-responsive element, certain oriLyt sequences required for maximal BMRF1-responsiveness were not required for maximal Sp1-responsiveness. In particular, a site-directed mutation altering the downstream component sequence GATGG (located from -588 to -592 relative to the BHLF1 transcription initiation site) did not affect Sp1-responsiveness, but reduced BMRF-1-responsiveness by 75% and abolished oriLyt replication. Although BMRF1 possesses nonspecific DNA binding activity, were unable to demonstrate specific BMRF1 binding to the downstream component of oriLyt. Our results suggest that BMRF1-induced activation of the essential downstream component of oriLyt may play an important role in oriLyt replication.