Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus implicated in lymphomas, such as Burkitt's lymphoma, Hodgkin's lymphoma, and NK/T cell lymphoma. MicroRNAs (miRNAs) are 19-25 nucleotide long single-stranded RNAs involved in post-transcriptional gene regulation. miRNAs are mainly transcribed by RNA polymerase II (pol II) to have stem-loop structures and subsequently processed by Drosha and Dicer. EBV miRNAs are expressed in B cells, nasopharyngeal carcinoma cells, and gastric carcinoma cells infected with EBV. EBV miRNAs can be divided into two groups: BHRF1 miRNAs and BART miRNAs. In this study, we investigated the biogenesis of EBV miRNAs. Treatment of the SNU-719 EBV-positive gastric cancer cell line with α-amanitin at a concentration that selectively inhibits RNA polymerase II activity decreased the expression levels of BART miRNAs. The expression levels of BART miRNAs were also reduced by RNA interference targeting Drosha and Dicer. Two of each C/EBPβ and c-Myc binding sites are located upstream of the proposed initiation sites for primary BART miRNA transcripts. Knockdown of C/EBPβ but not c-Myc using siRNAs reduced BART miRNA expression by 25-55% compared with the control. These results suggest that BART miRNAs are transcribed by pol II and undergo a similar biogenesis process with cellular miRNAs.