The NF-κB family members p65 (RelA) and c-Rel recognize similar DNA sequences, yet the phenotypes of mutant mice suggest that these proteins regulate distinct sets of genes. Here we demonstrate that 46 unique residues within an 86-residue segment of the Rel homology region (RHR) of c-Rel are responsible for the c-Rel requirement for Il12b gene induction by lipopolysaccharide in bone marrow-derived macrophages. These same residues were responsible for the c-Rel requirement for Il12a induction in dendritic cells, and in both instances, no evidence of c-Rel-specific coactivator interactions was found. Although the residues of c-Rel and p65 that contact specific bases and the DNA backbone within nuclear factor-κB (NF-κB) recognition sequences are identical, homodimers of c-Rel and of a chimeric p65 protein containing the critical c-Rel residues bound with high affinity to a broader range of NF-κB recognition sequences than did wild-type p65 homodimers. These results demonstrate that the unique functions of closely related transcription factor family members can be dictated by differences in the range of DNA sequences recognized at high affinity, despite having similar binding site consensus sequences and DNA contact residues.