NF-_B is a family of eukaryotic transcription factor proteins that inducibly regulates the expression of a large number of genes that play important roles in innate immunity and inflammation. NF-_B consists of varying homo- and heterodimers, one form of which is a homodimer of p50 subunits (p50:p50). The NF-_B p50 homodimer is unique among NF-_B proteins in that it escapes regulation through association with classical I_B proteins in the cytoplasm and enters the nucleus where it binds to DNA. In the nucleus, p50:p50 is the preferred binding partner of the "nuclear I_B" protein I_B_, itself an NF-_B responsive protein that is required for the further NF-_B-dependent expression of the pluripotent cytokine interleukin-6 (IL-6). This raises the possibility that ternary complexes of NF-_B p50 homodimer with I_B_ on target gene DNA could serve as a necessary signal for elevated expression of vital pro-inflammatory NF-_B target genes. Previously determined x-ray crystal structures of p50:p50 on _B DNA have revealed that this dimeric transcription factor binds to double-stranded DNA with variable spacing. We recently crystallized and determined the x-ray crystal structure of the DNA binding Rel homology region of murine p50 homodimer to _B DNA from the promoter of the IL-6 and NGAL genes and found, once again, that p50 binds the two sites with distinct spacing. In an attempt to identify the "preferred" binding mode of p50 on _B DNA, we prepared oligonucleotides with an "idealized" sequence _B DNA designed to allow p50:p50 to bind with 9-12 base pair spacing. The 3.0 Â x-ray co-crystal structure of the complex suggests that p50:p50 binds _B DNA with 11 base pair spacing when afforded freedom to select its ideal binding mode. We conclude that this is the preferred spacing of p50:p50 on _B DNA.