We have demonstrated in transient expression assays that the Epstein-Barr virus (EBV) DNA polymerase transactivates expression of the bacterial chloramphenicol acetyltransferase (CAT) gene linked to the human immunodeficiency virus (HIV) type 1 5' long terminal repeat (LTR). The evidence was provided by two sets of experiments. Transfection of Raji cells with HIV LTR-CAT followed by superinfection with EBV resulted in a 150-fold increase in CAT activity. In the presence of viral DNA inhibitor 3'-azido-3'-deoxythymidine (AZT), the CAT activity was inhibited by approximately 70%, suggesting that EBV DNA polymerase was involved in the transactivation of HIV LTR. The direct proof came from the cotransfection of HIV LTR-CAT with expression plasmid containing EBV polymerase gene; depending on the polymerase gene construct cotransfection with both plasmids resulted in a 23- to 38-fold increase of HIV LTR-CAT activity. The interaction between EBV polymerase and HIV may contribute to the role of EBV as a cofactor in the pathogenesis of AIDS.