The only member of the Epstein-Barr virus family of nuclear proteins (EBNAs) expressed during type I and type II latent infections is EBNA-1. This is in contrast to type III latency, during which all six nuclear proteins are expressed from a common transcription unit. The exclusive expression of EBNA-1 during type I and II latency is mediated through a recently identified promoter, Fp. The objective of this study was to characterize Fp in the Burkitt lymphoma cell background, where it is known to be differentially utilized. Using a short-term transfection assay and reporter gene plasmids containing Fp linked to the human growth hormone, we examined Fp activity in type I and type III latently infected and virus-negative Burkitt lymphoma cells. The data suggested that Fp is predominantly regulated through two distinct elements located between +24 and +270 relative to the transcription start site. One element positively mediates Fp activity, probably at the level of transcription, and acts in a virus-independent manner. The second element contains the EBNA-1 DNA binding domain III and negatively regulates Fp-directed gene expression in trans with EBNA-1 in type III as well as type I latency. Thus, we have identified a third function of EBNA-1, i.e., that of a repressor of gene expression, in addition to its known role in viral DNA replication and its ability to trans-activate gene expression. The overall activity of Fp in type I latently infected Burkitt cells was approximately sixfold lower than in virus-negative Burkitt cells, in which there is no autoregulation, suggesting that there is a fine balance between these two opposing regulatory elements during type I latency.