EBV infection of human B lymphocytes induces expression of the low affinity IgE receptor, Fc epsilon RII/CD23. CD23 is constitutively expressed in EBV-immortalized B cells and may play an essential role in immortalization. We previously explored the regulation of CD23 by EBV, showing that induction results from transcriptional activation that is mediated, in part, by an EBV-responsive transcriptional regulatory element in the 5' region of CD23 (-229 to +305 relative to the type a promoter). We now report the localization of the regulatory element and characterization of its cognate DNA-binding proteins. Reporter gene assays in EBV-positive and -negative lines localized a functional EBV-responsive enhancer to a 37-bp fragment (+248 to +284) that contains a GC-rich sequence (GC box) within intron I of type a CD23. This fragment was shown by mobility shift assays to specifically bind nuclear protein(s) from EBV-positive lines, but not EBV-negative lines. Mutation of the GC box resulted in a loss of protein-binding activity, implicating involvement of a GC box-binding protein in the DNA/protein interaction. Supershift assays suggested that the ubiquitous GC box-binding transcription factor, Sp1, is not a part of the complex, and UV-crosslinking studies demonstrated that the DNA/protein complex contains at least two proteins that differ in size from other known GC box-binding proteins. Binding of these proteins to the enhancer element requires phosphorylation, because phosphatase treatment of nuclear extracts abolished formation of the DNA/protein complex. These studies reveal the presence of an EBV-responsive enhancer element in intron I of type a CD23 and implicate a GC box-binding transcription factor in the activation of CD23 by EBV.