We have applied bioinformatic analysis of X-ray 3D structures of complexes of transcription factor NF-κB with DNAs. We determined the number of possible Van der Waals contacts and hydrogen bonds between amino acid residues and nucleotides. Conservative contacts in the NF-κB dimer-DNA complex composed of p50 and/or p65 NF-κB subunit and DNA sequences like 5′-GGGAMWTTCC-3′ were revealed. Based on these results, we propose a novel scheme for interactions between NF-κB p50 homodimer and the κB region of the immunoglobulin light chain gene enhancer (Ig-κB). We applied a chemical cross-linking technique to study the proximity of some Lys and Cys residues of NF-κB p50 subunit with certain reactive nucleotides into its recognition site. In all cases, the experimentally determined protein-DNA contacts were in good agreement with the predicted ones.