Epstein-Barr virus (EBV) has been associated with human cancers of lymphocytic or epithelial origin. Potential functions of the BARF1 early gene in EBV oncogenesis emerged from our observations showing expression of BARF1-encoded protein in nasopharyngeal carcinoma biopsies, and induction of either malignant transformation (in rodent fibroblast and human B cell lines) or immortalization (in monkey primary epithelial cells) following BARF1 transfection. We previously reported expression of the BARF1 product as a cytoplasm/membrane-associated protein from 293-tTA cells infected with a BARF1-recombinant adenovirus. Since constitutive expression of BARF1 from this heterologous system became inefficient, we developed a tetracycline-regulatable recombinant vector expressing BARF1 and green fluorescent protein from a dicistronic message. As here reported, stable and efficient expression of BARF1 from this vector in either permissive or non-permissive cell lines, allowed the first sequencing identification and further molecular characterization of BARF1-encoded protein.