Analysis by electrophoretic mobility shift assays (EMSA) of the different proteins associated with the κB sequence of the interleukin-6 (IL-6) promoter (IL6-κB) allowed us to detect a specific complex formed with the recombination signal sequence binding protein Jκ (RBP-Jκ). Single-base exchanges within the oligonucleotide sequence defined the critical base pairs involved in the interaction between RBP-Jκ and the IL6-κB motif. Binding analysis suggests that the amount of RBP-Jκ protein present in the nucleus is severalfold higher than the total amount of inducible NF-κB complexes but that the latter bind DNA with a 10-fold-higher affinity. A reporter gene study was performed to determine the functional implication of this binding; we found that the constitutive occupancy of the IL6-κB site by the RBP-Jκ protein was responsible for the low basal levels of IL-6 promoter activity in L929sA fibrosarcoma cells and that RBP-Jκ partially blocked access of NF-κB complexes to the IL-6 promoter. We propose that such a mechanism could be involved in the constitutive repression of the IL-6 gene under normal physiological conditions.