The experiments carried out in this study in MyD88-, TRIF-, Unc93B and various TLR-deficient mice excluded a participation of TLRs in the IFN-ab responses to Ads, including the recombinant Ad5-GFP. The only exception was the TLR9-and MyD88-dependent IFN-ab response elicited by Ad2Ts1, a mutant virus deficient in endosomal escape [5] . However, compared to all other Ads used in this study, Ad2Ts1 induced very low levels of IFN-ab, with faster kinetics and only when used in very high amounts. This is consistent with the finding that the mechanisms of type I IFN induction by Ad2Ts1 and the other adenoviruses are not the same. We suggest that the fast escape of Ads from the endosome circumvents activation of endosomal TLRs. The requirement for a longer endosomal retention time in TLR9dependent IFN-ab production has been recently demonstrated [82] . On the whole, our experiments indicate that Ad endosomal escape is required for the induction of IFN-ab in vivo and suggest a cytosolic pathway. The same requirement was ascertained in the present study for the IL-6 response.