Adenoviruses (Ads) are important pathogens and promising vectors for gene therapy applications. In the course of adenoviral infections innate immune responses are activated, which can be beneficial for the antiviral host defense but also detrimental if activated in a deregulated manner. Type I IFNs are crucial for the innate immune control of various viral infections in the mammalian host. So far, the early, systemic release of IFN-ab during viral infections has been attributed to specialized immune cells, the plasmacytoid dendritic cells. Here, in a mouse infection model, we show that wild type Ads, as well as adenoviral vectors, elicit rapid IFN-ab production almost exclusively in another cell population, the splenic myeloid dendritic cells. This IFN-ab storm depends on viral escape from endosomes to the cytosol and the requirements of the response are suggestive of a novel viral induction pathway. Furthermore, we show that virus induced IFN-ab is the key mediator of Ad-induced hypersensitivity to the cytokine-inducing and toxic activity of lipopolysaccharide, a common constituent of Gram-negative bacteria. Since these bacteria comprise several commensals and pathogens, enhanced susceptibility to lipopolysaccharide may contribute to toxic reactions observed during adenoviral gene therapy and to the clinical symptoms of adenoviral diseases. Titration of the viral preparations in mice revealed that a positive correlation between the viral dose and the height of the IFN-ab response existed only when relatively small doses of intact Ads were used. Higher doses of the Ads either did not elicit a further increase of the IFN-ab response (Ad3, Fig. 1B ) or led to a decrease of the response (Ad R700, Fig. S2C ). In contrast, injection of the corresponding UV-inactivated Ads always led to a gradual increase of the IFN-ab response and, at higher viral concentrations, even exceeded the response obtained with intact viruses. Thus, the expression of Ad genes is not required for the induction of IFN-ab in mice. The data also indicate that the expression of early adenoviral genes negatively regulates type I IFN production. Notably, the Ad E1A gene has been shown previously to suppress Newcastle Disease Virus and IRF-3 induced IFN-a4 promoter induction in transient expression assays in fibroblasts [43] .