We tested whether compounds related to c-di-GMP were capable of stimulating IFN- expression when delivered to the cytosol. We measured induction of endogenous type I IFN protein by bioassay ( Fig. 1 B) , and induction of IFN- and IFN-5 mRNA levels by quantitative RT-PCR ( Fig. 1 , C and D). No detectable IFN- production was observed upon transfection with GTP, cGMP, ppGpp (a different bacterial nucleotide second messenger), or pGpG (hydrolyzed c-di-GMP), whereas the cytosolic response to 3.3 µg/ml of transfected c-di-GMP was of a similar magnitude to that of 3.3 µg/ml of transfected DNA (pdA:dT) and RNA (pI:C), which are known cytosolic inducers of type I IFN. We do not believe that the cytosolic induction of type I IFNs in response to c-di-GMP is caused by a contaminant (such as LPS) because a role in IFN--mediated antiviral immunity (Tenoever et al., 2007) , and found that Ikbke / macrophages responded normally to c-di-GMP (Fig. 2 C) .