In order to characterize the induction of type I IFNs by Ad in vivo, we first examined how two types of human Ad, Ad3 (species B) and Ad R700 (species C) [2] known to use distinct infectious entry routes, elicit an IFN-ab response in vivo. The results summarized in Fig. 1A and supplementary Fig. S1A show that all mice infected with either of the two viruses exhibited similar IFN-ab responses. IFN-ab was first detectable in plasma at 4 h, peaking at 8 h and declining to low levels 18 h after infection. We then investigated, whether the expression of viral genes is required and/or regulate the induction of IFN-ab by Ads. To this end, we injected mice with an UV-inactivated Ad3 (Fig. S1B ) or Ad R700, incapable of viral gene expression, or with a recombinant Ad5 (species C) that contains a deletion of the E1 and E3 Ad early regions and expresses GFP (Ad5-GFP) (Fig. S1C ). As shown in Fig. 1A , UV-inactivated Ads also induced a strong IFN-ab response which, however, peaked at 6 h after injection, i.e. 2 h earlier than the response to intact Ads. A similar early-peaking IFN-ab response was obtained in mice injected with UV inactivated Ad R700 (Fig. S2A ) and with the recombinant Ad5-GFP (Fig. S2B ).