It has been well established that innate immune responses are initiated in response to certain microbial ligands that are evolutionarily conserved and that can be distinguished from selfligands. Nucleic acids appear to be a favored target of immune recognition, and are sensed by a variety of endosomal and cytosolic sensor proteins. The cyclic dinucleotide c-di-GMP is a bacterial second messenger that exhibits several characteristics that are desirable in an immunostimulatory ligand: it is produced by numerous species of bacteria, it is a critical regulator of bacterial physiology, and it is not similar to host molecules. Indeed, several previous reports have demonstrated that c-di-GMP can provoke potent immune responses when injected in vivo into mice (Karaolis et al., 2005a; Karaolis et al., 2005b; Karaolis et al., 2007a; Karaolis et al., 2007b) . However, the mechanism by which c-di-GMP stimulates immune responses remained unclear. Our data provide evidence that c-di-GMP is sensed by a novel cytosolic immunosurveillance pathway. Responsiveness to c-di-GMP is independent of TLRs that monitor the extracellular/endosomal compartments and is strongly potentiated by transfection of c-di-GMP into the host cell cytosol. Moreover, c-di-GMP provokes a transcriptional response highly reminiscent of that triggered by the cytosolic presence of DNA or RNA, and involves activation of TBK-1, MAP kinases, and the IRF-3 and NF-B transcription factors. Thus, we propose that c-di-GMP is sensed in the cytosol.