Approximately 10% of gastric adenocarcinomas worldwide are associated with human EBV. These carcinomas generally do not express the latent membrane protein 1 (LMP1), the major known EBV oncogene. Recently, another EBV gene [ie., BARF1 (BamHI A rightward open reading frame)] was shown to have transforming and immortalizing capacities. Therefore, in this study, we investigated the expression of BARF1 in EBV-carrying gastric adenocarcinomas in relation to the expression of other latent EBV transcripts. In the present study, 10 of 132 gastric adenocarcinomas tested positive for EBV using EBER1/2-RNA in situ hybridization. We demonstrate BARF1 gene transcription in nine EBV-carrying gastric adenocarcinomas (with sufficient RNA quality) using the BARF1-specific nucleic acid sequence-based amplification assay. In addition, we also detected other latent EBV transcripts (ie., BARF0-, LMP2A-, and Q/K-driven EBNA1 transcripts in these carcinomas using reverse transcription-PCR analysis. No expression of LMP1, EBNA2, and ZEBRA (either at transcription or protein level) was found. In addition, two cases were positive for BHRF1 transcripts, the viral bcl-2 homologue. Thus, together with BARF1 transcription, a unique and distinct EBV latency type has been found in EBV-associated gastric adenocarcinomas. Because BARF1 exerts immortalizing effects on human epithelial cells in vitro and EBV-carrying gastric adenocarcinomas lack the expression of LMP1, the BARF1 gene might act as the viral oncogene in EBV-carrying gastric carcinomas. The BARF1 gene offers an alternative way for EBV-mediated oncogenesis other than LMP1.