Only EBV-infected B lymphocytes which express the B cell activation marker CD23 are capable of establishing immortalised cell lines. Epstein-Barr virus determined nuclear antigen 2 (EBNA-2) is essential for EBV-induced growth transformation (Cohen, et al, 1989) and can specifically upregulate CD23 gene expression (Wang, et al, 1987). The regulation of CD23 gene expression is likely to be an important factor in EBV-induced B cell growth transformation. The cellular CD23 promoter and EBV TP-1 promoter have previously been shown to be transactivated by EBNA-2 (Wang, et al, 1991, Le Roux, et al, 1993). Studies have also shown that while EBNA-3A itself has no effect on the TP-1 promoter, it can repress EBNA-2-mediated transactivation in a B cell background (Le Roux, et al, 1994). Homologies have been shown between sequences present in the EBV TP-1 EBNA-2 responsive element and other functionally similar sequences which are found in the CD23 promoter region. The purpose of this study was to explore the regulation of CD23 gene expression by the EBV latent proteins EBNA-2 and EBNA-3A. The most common reporter gene used in transfection studies is CAT (chloramphenicolacetyl transferase), usually assayed using a radioisotopic method. To obviate the need to use a radioassay, an alternative reporter plasmid was constructed and tested in a range of cell backgrounds. This construct consisted of the CD23 promoter cloned upstream of the PAP gene (placental alkaline phosphatase). The assay involves a colorimetric reaction, and is a simpler and cheaper reporter assay (Henthorn, et al, 1988). In order to identify relevant sequence modules in the CD23 promoter, deletion mutants of CD23CAT were constructed for use in co-transfection assays with plasmids encoding EBNA-2/3A. A strategy based on using the nuclease BAL 31 was designed to create nested mutations. Fragments of the CD23 promoter including the EBNA-2 responsive elements were also amplified by PCR for cloning upstream of CAT.