The location of the BcLF1 promoter of Epstein-Barr virus (EBV) has been identified by primer extension, which indicates that the +1 site of the BcLF1 mRNA is located at nucleotide 137676 of the EBV genome. According to deletion analysis, the region upstream from nucleotide -38 is not essential for transcription of BcLF1. A 23 bp region in the promoter, from nucleotide -38 to -16, was identified as necessary for regulating the expression of BcLF1, i.e. the promoter activity is activated by 12-O-tetradecanoylphorbol 13-acetate but is repressed by phosphonoacetic acid. The results presented also demonstrate that the oriLyt sequence in cis is essential for enhancing the expression of BcLF1.