The innate immune response in Drosophila involves the inducible expression of antimicrobial peptide genes mediated by the Toll and IMD signaling pathways. Dorsal and DIF act downstream of Toll, whereas Relish acts downstream of IMD to regulate target gene expression. Dorsal, DIF, and Relish are NF-kappaB-related transcription factors and function as obligate dimers, but it is not clear how the various dimer combinations contribute to the innate immune response. We systematically examined the dimerization tendency of these proteins through the use of transgenic assays. The results show that all combinations of homo- and heterodimers are formed, but with varying degrees of efficiency. The formation of the DIF-Relish heterodimer is particularly interesting because it may mediate signaling for the seemingly independent Toll and IMD pathways. By incorporating a flexible peptide linker, we specifically tested the functions of the DIF;Relish (a ; sign represents the peptide linker) linked heterodimer. Our results demonstrate that the linked heterodimer can activate target genes of both the Toll and IMD pathways. The DIF and Relish complex is detectable in whole animal extracts, suggesting that this heterodimer may function in vivo to increase the spectrum and level of antimicrobial peptide production in response to different infections.