Epstein-Barr virus (EBV) nuclear protein 2 (EBNA-2) is essential for B-lymphocyte growth transformation. EBNA-2 upregulates mRNAs encoding CD23, a B-lymphocyte surface protein closely associated with EBV-induced growth transformation. To further investigate this EBNA-2 effect, we searched in the genomic DNA spanning the type a and type b CD23 mRNA start sites for a cis-acting fragment that would render a promoter transactivatable by EBNA-2. An 800-bp CD23 DNA fragment (-335 to +465 relative to the type a CD23 mRNA start site) conferred EBNA-2 responsiveness to the herpes simplex virus thymidine kinase (TK) promoter when transfected into EBV-negative B-lymphoma cells. Deletional analysis identified a -275/-89 subfragment that was EBNA-2 responsive when cloned in either orientation and at variable distances upstream of the heterologous promoter. EBNA-2 and the cis-acting CD23 element increased TK-promoted mRNA and did not alter the herpes simplex virus TK promoter transcription start site. As expected, a type a CD23 promoter (-335/+80) which contained the EBNA-2-responsive element was transactivated by EBNA-2. As in EBV infection and stable EBNA-2 transfection, the CD23 DNA element in cis with heterologous or homologous promoters was less responsive to type 2 than to type 1 EBNA-2, whereas the EBNA-2-responsive DNA fragment from the EBV latent membrane protein 1 promoter was more responsive to the type 2 EBNA-2. These experiments delineate a 186-bp, EBNA-2-responsive cell DNA fragment and provide firm evidence that EBNA-2 transactivates transcription of cell genes. The greater type 1 versus type 2 EBNA-2 responsiveness of the CD23 promoter and the lack of a similar effect on the latent membrane protein 1 promoter is consistent with the hypothesis that greater cell gene transactivation by type 1 EBNA-2 is the basis for the more efficient growth-transforming properties of type 1 EBV.