The induction of type I IFN in Ad-infected mice has been recently studied [10, 42] and associated with both the extracytoplasmic and intracytoplasmic pathways. It was claimed that a part of the IFN-ab response is initiated by TLR9 and MyD88 signaling in pDCs and another part by cytosolic DNA recognition in non-pDCs. However, the identification of IFN-ab producing cell types directly in infected mice was not carried out. In the present study we investigated the IFN-ab responses of Ad-infected mice and showed that the bulk of the in vivo induced IFN-ab is produced by splenic mDCs. Furthermore, we found that TLRs, including TLR9 play no major role. The Ad-elicited IFN-ab response required viral endosomal escape, suggesting a cytosolic induction pathway. Surprisingly however, the induction was independent of IRF-3 and dependent on stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) activity, which is in contrast to the known induction mechanism initiated through cytosolic DNA recognition. Instead, the induction required IRF-7, and a positive feedback regulation via the type I IFN receptor. Although this does not exclude a role for cytosolic nucleic acid sensors, our data do not support the involvement of MDA-5, RIG-I and Zbp1/DAI in the induction of the IFN-ab response to Ad. Furthermore, our results reveal distinct mechanisms in the induction of IFN-ab and IL-6 by Ad. Finally, we show that Ad-induced IFN-ab is a key mediator of hypersensitivity to bacterial lipopolysaccharides in infected mice. Enhanced susceptibility to LPS and to other microbial inducers of inflammation may contribute to toxic reactions observed during adenoviral gene therapy and to the clinical symptoms of adenoviral diseases.