NF-κB2 (p100/p52), a member of the NF-κB/Rel family of transcription factors, is involved in the regulation of a variety of genes important for immune function. Previously, we have shown that the NF-κB2 gene is regulated in a positive and a negative manner. Two κB elements within the NF-κB2 promoter mediate tumor necrosis factor alpha-inducible transactivation. In addition, we have shown that there exists a transcriptional repression in the absence of NF-κB. To identify a DNA binding activity responsible for this transcriptional repression, we have partially purified a nuclear complex, named Rep-κB. Here we further analyze this putative repressive binding activity. Detailed examination of Rep-κB–DNA interaction revealed the sequence requirements for binding to be almost identical to those of recombination signal binding protein Jκ (RBP-Jκ), the mammalian homolog of the protein encoded by Drosophila suppressor of hairless [Su (H)]. In addition, in electromobility shift assays, Rep-κB binding activity is recognized by an antibody directed against RBP-Jκ. By performing transient-transfection assays, we show that human RBP-Jκ represses basal as well as RelA (p65)-stimulated NF-κB2 promoter activity. Studies in Drosophila melanogaster have shown that Su (H) is implicated in the Notch signaling pathway regulating cell fate decisions. In transient-transfection assays we show that truncated Notch-1 strongly induces NF-κB2 promoter activity. In summary, our data clearly demonstrate that Rep-κB is closely related or identical to RBP-Jκ. RBP-Jκ is a strong transcriptional repressor of NF-κB2. Moreover, this repression can be overcome by activated Notch-1, suggesting that NF-κB2 is a novel putative Notch target gene.