The phenobarbital-inducible rat cytochrome P450 (CYP) 2B1 and 2B2 proteins are encoded by homologous genes whose promoters contain a mammalian-apparent long terminal repeat retrotransposon (MaLR). An NF-kappaB-like site within the MaLR forms multiple protein-DNA complexes with rat liver and HeLa cell nuclear extracts. Using antibody supershift assays, we have identified these complexes as NF-kappaB and RPB-Jkappa/CBF1. Competition assays using a series of single site mutant oligonucleotides reveal that the recognition sites for these two factors overlap. We also show that the CYP2B1/2 NF-kappaB element, but not the Igkappa NF-kappaB element, can repress transcription in vitro when positioned upstream of the heterologous adenovirus major late core promoter. In addition, RBP-Jkappa over-expressed in COS-7 cells repressed expression in vivo from an SV40-luciferase reporter construct that contained the CYP2B1/2 NF-kappaB element. Finally, we observe similar levels of NF-kappaB and RBP-Jkappa binding activities in nuclear extracts prepared from control and phenobarbital-induced rat livers. The results suggest that RBP-Jkappa/CBF1 binds an atypical NF-kappaB site in the CYP2B1/2 promoters and may help to maintain a low level of expression in the absence of inducer.