We hypothesized that c-di-GMP might be sensed by a cytosolic sensor leading to the induction of type I IFNs. To test this hypothesis, we compared the ability of overlayed versus transfected c-di-GMP to induce type I IFN in bone marrow macrophages. At the concentrations used (up to 25 µg/ml), overlay of c-di-GMP did not elicit a significant response ( Fig. 1 A) , although IFN- production could be detected at higher concentrations (50 or 100 µg/ml; not depicted). In contrast, delivery of c-di-GMP to the cytosol by transfection elicited robust activation of IFN- in a dose-dependent manner ( Fig. 1 A) . A significant response was detected with a concentration of c-di-GMP as low as 2.5 µg/ml (equivalent to 3.6 µM). These results suggested that c-di-GMP signals via a cytosolic immunosurveillance pathway.