Two sequences required for activity of the Epstein-Barr virus BART RNA promoter in transfection assays have been identified by site-directed mutagenesis. One contains a consensus AP-1 site; the other has some similarity to Ets and Stat consensus binding sites. Candidate sequences were suggested by mapping a region of unmethylated DNA in EBV around the BART promoter followed by in vivo footprinting the promoter in the C666-1 nasopharyngeal carcinoma cell line, which expresses BART RNAs. The data are presented in the context of a revised EBV DNA sequence, known as EBV wt, that is proposed as a future standard sequence for EBV.