NF-kappaB2 (p100/p52), a member of the NF-kappaB/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-kappaB2 gene is regulated in a positive and a negative manner. Two kappaB elements within the NF-kappaB2 promoter mediate tumor necrosis factor alpha-inducible transactivation. In addition, we have shown that there exists a transcriptional repression in the absence of NF-kappaB. To identify a DNA binding activity responsible for this transcriptional repression, we have partially purified a nuclear complex, named Rep-kappaB. Here we further analyze this putative repressive binding activity. Detailed examination of Rep-kappaB-DNA interaction revealed the sequence requirements for binding to be almost identical to those of recombination signal binding protein Jkappa (RBP-Jkappa), the mammalian homolog of the protein encoded by Drosophila suppressor of hairless [Su(H)]. In addition, in electromobility shift assays, Rep-kappaB binding activity is recognized by an antibody directed against RBP-Jkappa. By performing transient-transfection assays, we show that human RBP-Jkappa represses basal as well as RelA (p65)-stimulated NF-kappaB2 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-kappaB2 promoter activity. In summary, our data clearly demonstrate that Rep-kappaB is closely related or identical to RBP-Jkappa. RBP-Jkappa is a strong transcriptional repressor of NF-kappaB2. Moreover, this repression can be overcome by activated Notch-1, suggesting that NF-kappaB2 is a novel putative Notch target gene.