Infection with Epstein-Barr virus (EBV) usually leads to a latent state in B lymphocytes. The virus can be reactivated through two viral transactivators. Zta and Rta, leading to a cascade of gene expression. An EBV DNA array was generated to analyze the pattern of transcription of the entire EBV genome under various Conditions. Firstly, a complete set of temporal expression clusters of EBV genes was displayed by analyzing the array data of anti-IgG-induced Akata cells. In addition to assigning genes of unknown function to the various Clusters, increasing expression of latent genes, including EBNA2, EBNA3A and EBNA 3C, was observed during virus replication. Secondly, gene expression independent of viral DNA replication was analyzed in PAA blocked Akata cells and in chemically induced Raji cells. Several genes with presumed late functions were found to be expressed with early kinetics and independent of vital DNA replication, suggesting possible novel functions for these genes. Finally, the EBV array was used to identify Rta responsive gene expression in Raji cells, and in the EBV-positive epithelial cells NA, using a Zta siRNA strategy. The array data were confirmed by Northern blotting, RT-PCR and reporter assays. All the information here thus provides a better understanding of the control of EBV lytic gene expression. (c) 2005 Elsevier Inc. All rights reserved.