In every latently Epstein-Barr virus-infected cell the viral genes EBER-1 and EBER-2 are transcribed by polymerase III. In lytically infected cells in vivo the EBER genes could not be detected. However, in cell culture downregulation could not be confirmed, and hence the relevance of this shutdown to the replication of the virus was not clear. We assayed the transcriptional activity of the EBER genes by nuclear run-on assays with enriched lytically infected cells and demonstrated that EBER-1 and EBER-2 are differentially downregulated on the transcriptional level during the switch to lytic viral replication. This downregulation was an early event during the lytic replication of the virus.