The safety of gene therapy vectors is a major concern when novel viral or nonviral therapeutics are proposed for applications in humans. Adenovirus (Ad) vectors have been extensively used as efficient gene delivery vehicles in vitro over the last two decades. However, upon i.v. application, they elicit robust innate and inflammatory responses that may be fatal for the host. To date, the primary cytokines and chemokines involved in the initiation of these host responses remain illusive. In this study, we demonstrate that IL-1 is a major mediator involved in the initiation of immediate host responses toward i.v. applied Ad vectors. Using mice in which IL-1 signaling was genetically eliminated (IL-1RI-KO), or wild-type animals for which signaling was blocked by anti-IL-1 Abs, we found that i.v. applied Ad vectors elicited dramatically reduced acute inflammatory responses when compared with control animals. Importantly, the efficiency of Ad gene transfer in vivo was not significantly affected by interfering with IL-1 signaling. Using an in situ hybridization technique, we found that hepatocytes and Kupffer cells trigger IL-1 transcription in liver tissue after i.v. Ad vector administration. We also found that expression of the MIP-2 chemokine gene (which is responsible for recruitment of neutrophils to the liver) depends on IL-1 activation. Our data indicate that immediate innate and inflammatory host responses toward i.v. applied Ad vectors can be pharmacologically controlled through interference with IL-1 signaling pathways.