Expression of human complement receptor type 2 (CR2/CD21) is primarily restricted to mature B cells and follicular dendritic cells. We previously described an intronic transcriptional silencer that controls the appropriate B cell-specific and developmentally restricted expression of human CR2/CD21 in both stably transfected cell lines and transgenic mice. Here we report the identification of a nucleotide sequence within the 2.5 kb CR2 silencer (CRS) that is crucial to its silencer function. This site comprises a binding site for the transcriptional repressor CBF1 (RBP-J or RBP-Jkappa) as well as Sp1 and other as yet uncharacterized proteins. A 2-bp mutation which eliminates the binding of CBF1 and other protein(s) in vitro results in loss of silencer activity in vivo. These results demonstrate the importance of this site in regulating CR2 expression and suggest that CBF1, a component of the developmentally important Notch signaling pathway, may play a role in the control of human CR2 gene expression.