NF-κB and C/EBP represent distinct families of transcription factors that target unique DNA enhancer elements. The heterodimeric NF-κB complex is composed of two subunits, a 50- and a 65-kDa protein. All members of the NF-κB family, including the product of the proto-oncogene c-rel, are characterized by their highly homologous ~300-amino-acid N-terminal region. This Rel homology domain mediates DNA binding, dimerization, and nuclear targeting of these proteins. C/EBP contains the bZIP region, which is characterized by two motifs in the C-terminal half of the protein: a basic region involved in DNA binding and a leucine zipper motif involved in dimerization. The C/EBP family consist of several related proteins, C/EBPα, C/EBPβ, C/EBPγ, and C/EBPδ, that form homodimers and that form heterodimers with each other. We now demonstrated the unexpected cross-coupling of members of the NF-κB family three members of the C/EBP family. NF-κB p65, p50, and Rel functionally synergize with C/EBPα, C/EBPβ, and C/EBPδ. This cross-coupling results in the inhibition of promoters with κ B enhancer motifs and in the synergistic stimulation of promoters with C/EBP binding sites. These studies demonstrate that NF-κB augments gene expression mediated by a multimerized c-fos serum response element in the presence of C/EBP. We show a direct physical association of the bZIP region of C/EBP with the Rel homology domain of NF-κB. The cross-coupling of NF-κB with C/EBP highlights a mechanism of gene regulation involving an interaction between distinct transcription factor families.